Bryostatin analogues, synthetic methods and uses

ABSTRACT

Biologically active compounds related to the bryostatin family of compounds, including methods of utilizing the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/967,000, filed Aug. 31, 2007, which is incorporated herein by reference in its entirety.

FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contract CA031845 awarded by the National Institutes of Health. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention concerns biologically active compounds related to the bryostatin family of compounds, and to methods and compositions utilizing them.

BACKGROUND

Protein kinase C (PKC) related disorders are important causes of illness, disability and death worldwide, and represent important therapeutic targets. The broad range of disorders which are mediated by PKC include, for example, hyperproliferative diseases, immune related disorders, and cognitive disorders among others. There is need for new therapeutic agents which can target PKC to treat patients with these conditions.

SUMMARY OF THE INVENTION

One aspect of the present invention concerns bryostatin analogues, i.e., the compounds represented by Formula I:

In one aspect of the invention, a compound of Formula I is provided

wherein X is O, S or NR′, R³ is H or OH; R⁷ is selected from the group consisting of optionally substituted lower alkyl, optionally substituted alkenyl, hydroxyl, amino, optionally substituted alkylamino, ═O, optionally substituted acylamino, OC(O)NR′R′, OC(O)OR′, OC(O)R′, and substituted acyloxy; p is 0, 1, 2, 3, or 4;

R²⁰ is H, OH, or -T-U—V—R′ where T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent;

R²¹ is ═CR^(a)R^(b) or R²¹ represents independent moieties R^(c) and R^(d) where R^(a) and R^(b) are independently H, CO₂R′, CONR^(c)R^(d) or R′; R^(c) and R^(d) are independently H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3;

R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl and optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof, with the proviso that the compound is not

, wherein R″ is selected from the group consisting of CH₃, Ph, C₁₃H₂₇, C₇H₁₅, CH₂O(CH₂)₂O(CH₂)₂OCH₃, and

In some embodiments of the invention, a compound of Formula I is provided of the stereochemistry of Formula IA:

In some embodiments of the invention, a compound of Formula I is provided wherein X is O. In some embodiments of the invention, a compound of Formula I is provided wherein R³ is OH. In other embodiments, a compound of Formula I is provided wherein R²⁶ is H.

In another aspect of the invention, a compound of Formula II is provided

wherein R⁷ is selected from the group consisting of optionally substituted lower alkyl, optionally substituted alkenyl, hydroxyl, amino, optionally substituted alkylamino, ═O, optionally substituted acylamino, OC(O)NR′R′, OC(O)OR′, OC(O)R′, and substituted acyloxy; p is 0, 1, 2, 3, or 4;

R²⁰ is H, OH, or -T-U—V—R′ where T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent;

R²¹ is ═CR^(a)R^(b) or R²¹ represents independent moieties R^(c) and R^(d) where R^(a) and R^(b) are independently H, CO₂R′, CONR^(c)R^(d) or R′; R^(c) and R^(d) are independently H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3;

R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted aralkenyl, and optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof, with the proviso that the compound is not

wherein R″ is selected from the group consisting of CH₃, Ph, C₁₃H₂₇, C₇H₁₅, CH₂O(CH₂)₂O(CH₂)₂OCH₃, and

In some embodiments of the invention a compound of Formula II is provided of the stereochemistry of Formula IIA:

In some embodiments of the invention, a compound of Formula II is provided wherein R26 is H.

In a further aspect of the invention, a compound of Formula III is provided

wherein R³ is H or OH; R⁸ is selected from the group consisting of H, OH, R′, —(CH₂)O₂CR′, and —(CH₂)_(n)O₂C-haloalkyl; n is 0, 1, 2, 3, 4, or 5;

R⁹ is H or OH;

R²⁰ is H, OH, or -T-U—V—R′ where T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and

V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent;

R²¹ is ═CR^(a)R^(b) or R²¹ represents independent moieties R^(c) and R^(d) where R^(a) and R^(b) are independently H, CO₂R′, CONR^(c)R^(d) or R′; R^(c) and R^(d) are independently H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3;

R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted aralkenyl, and optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof, with the proviso that the compound is not

In some other embodiments of the invention, a compound of Formula II is provided having the stereochemical configuration of Formula IIIA:

In another aspect of the invention, a compound of Formula IV is provided

wherein R³ is H or OH;

R⁷ is selected from the group consisting of optionally substituted lower alkyl, optionally substituted alkenyl, hydroxyl, amino, optionally substituted alkylamino, ═O, optionally substituted acylamino, OC(O)NR′R′, OC(O)OR′, OC(O)R′, and substituted acyloxy; p is 0, 1, 2, 3, or 4;

R²⁰ is H, OH, or -T-U—V—R′ where T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent;

R²¹ is H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3;

R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted aralkenyl, and optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof.

In some embodiments of the invention, a compound of Formula IV is provided of the stereochemistry of Formula IVA:

In some of the embodiments of the invention, compounds of Formula IV are provided wherein R³ is OH. In some of the embodiments of the invention, compounds of Formula IV are provided wherein R²⁶ is H.

Exemplary compounds of the invention include, but are not limited to

and their pharmaceutically acceptable salts thereof.

In another aspect of the invention, a method of manufacturing a compound of Formula I, II, II or IV is provided comprising synthesizing said compound. Synthetic processes and methods are described herein.

In another aspect of the invention, a pharmaceutical composition is provided comprising a pharmaceutically effective amount of a compound of Formulas I, II, III or IV, or its pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable excipient. In some embodiments of the invention, the pharmaceutical composition comprises a pharmaceutically effective amount of a second agent having therapeutic activity via a mechanism distinct from that of the compound of Formula I, II, III, or IV. In other embodiments of the invention, the pharmaceutical composition further comprises a pharmaceutically effective amount of a second therapeutic agent having an immunosuppressive mechanism. In some embodiments of the invention, the pharmaceutical composition comprises more than one pharmaceutically acceptable excipient. In some embodiments of the invention, the pharmaceutical composition is administered via an oral, intravenous, intra-arterial, intramuscular, local, intraperitoneal, parenteral, transdermal, ocular, or intrathecal route.

In another aspect of the invention, a method is provided to treat a mammal suffering from a hyperproliferative cellular disorder or an immune-related disorder comprising administering a therapeutically effective amount of a compound of Formula I, II, III, or IV, or its pharmaceutically acceptable salts or esters thereof, to said mammal.

In a further aspect of the invention, a method is provided to treat a mammal suffering from a disorder related to protein kinase C activity comprising administering a therapeutically effective amount of a compound of Formula I, I, III or IV, or its pharmaceutically acceptable salts, to said mammal.

In another aspect of the invention, a method is provided to treat a cancerous cell comprising contacting a cancerous cell with a therapeutically effective amount of a compound of Formula I, I, III or IV, or its pharmaceutically acceptable salt or ester thereof, in an amount effective to inhibit growth or proliferation of said cell.

In some embodiments of the methods of the invention, the method further comprises the step of administering a therapeutically effective amount of a second agent having therapeutic activity via an immunosuppressive mechanism distinct from that of the compound of Formula I, II, III, or IV. In other embodiments of the methods of the invention, the method further comprises administering a therapeutically effective amount of a second agent having therapeutic activity via an immunosuppressive mechanism. In some embodiments of the methods of the invention, said administration of said second agent is before, after, or concomitantly with the administration of the compound of Formula I, II, III, or IV. In some embodiments of the method, administration of the compound of Formula I, II, III, or IV is via an oral, intravenous, intra-arterial, intramuscular, local, intraperitoneal, parenteral, transdermal, ocular, or intrathecal route. In some of the embodiments of the invention, the second agent is administered via the same route of administration as the compound of Formulas I, II, III or IV. In some embodiments of the method of the invention, the administration of the second therapeutic agent is via a different route of administration from the compound of Formula I, II, III, or IV.

In some of the methods of the invention, the hyperproliferative cellular disorders or immune related disorders include tumors and cancers; unwanted angiogenesis, psoriasis, blood vessel proliferation disorders, fibrotic disorders, autoimmune disorders, disorders brought about by abnormal proliferation of mesangial cells, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic micro-angiopathy syndromes, transplant rejection, and glomerulopathies, rheumatoid arthritis, ischemic heart disease, post-dialysis syndrome, leukemia, vasculitis; lipid histiocytosis, septic shock, inflammation, acute and chronic nephropathies, arterial restenosis, autoimmune diseases, and ocular diseases with retinal vessel proliferation. In some of the methods of the invention, the disorder related to protein kinase C activity inlcudes Alzheimer's disease, cognitive disorders and depression.

These and other objects and features of the invention will be better understood in light of the following detailed description.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Particularly, the complete disclosures of U.S. Pat. Nos. 6,624,189 and 7,256,286, both to Wender et al, are incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Protein kinase C modulators can be used to treat any one of the conditions which are mediated by PKC in order to benefit a patient. One application, amongst the wide range of disorders responsive to treatment by a PKC modulator is cancer.

Cancer, one of the hyperproliferative disorders mediated by PKC, is a major cause of death in the developed countries, with more than 500,000 human fatalities occurring annually in the United States. Cancers are generally the result of the transformation of normal cells into modified cells that proliferate excessively, leading to the formation of abnormal tissues or cell populations. Cancers have been observed for many different tissue and cell types, with cancers of the lung, breast, and colorectal system accounting for about half of all cases.

Currently, about one-third of cancer patients can be cured by surgical or radiation techniques. However, these approaches are most effective with cancerous lesions that have not yet metastasized to other regions of the body. Chemotherapeutic techniques currently cure another 17% of cancer patients. Combined chemotherapeutic and non-chemotherapeutic protocols can further enhance prospects for full recovery. Even for incurable cancer conditions, therapeutic treatments can be useful to achieve remission or at least extend patient longevity.

Numerous anticancer compounds have been developed over the past several decades. While these compounds comprise many different classes that act by a variety of mechanisms, one general approach has been to block the proliferation of cancerous cells by interfering with cell division. For example, anthracyclines, such as doxorubicin and daunorubicin, have been found to intercalate DNA, blocking DNA and RNA synthesis and causing strand scission by interacting with topoisomerase II. The taxanes, such as Taxol™ and Taxotere™, disrupt mitosis by promoting tubulin polymerization in microtubule assembly. Cis-platin forms interstrand crosslinks in DNA and is effective to kill cells in all stages of the cell cycle. As another example, cyclophosphamide and related alkylating agents contain di-(2-chloroethyl)-amino groups that bind covalently to cellular components such as DNA.

The bryostatins (Formula A) are a family of naturally occurring macrocyclic compounds originally isolated from marine bryozoa. Currently, there are about 20 known natural bryostatins which share three six-membered rings designated A, B and C, and which differ mainly in the nature of their substituents at C7 (OR^(A)) and C20 (R^(B)).

The bryostatins exhibit potent activity against a broad range of human cancer cell lines and provide significant in vivo life extensions in murine xenograft tumor models. Doses that are effective in vivo are extremely low, with activities demonstrated for concentrations as low as 1 μg/kg. Among additional therapeutic responses, the bryostatins have been found to promote the normal growth of bone marrow progenitor cells, provide cellular protection against normally lethal doses of ionizing radiation, and stimulate immune system responses that result in the production of T cells, tumor necrosis factors, interleukins and interferons. Bryostatins are also effective in inducing transformation of chronic lymphocytic leukemia cells to a hairy cell type, increasing the expression of p53 while decreasing the expression of bcl-2 in inducing apoptosis in cancer cells or at least pre-disposing a cell towards apoptosis, and reversing multidrug resistance (MDR).

At the molecular level, bryostatins have been shown to competitively inhibit the binding of plant-derived phorbol esters and endogenous diacyl glycerols to protein kinase C (PKC) at nanomolar to picomolar drug concentrations, and to stimulate comparable kinase activity. Unlike the phorbol esters, however, the bryostatins do not act as tumor promoters. Thus, the bryostatins appear to operate through a mode of action different from, and complementary to, the modes of action of established anticancer agents; such as cisplatin or taxol.

Further, their ability to bind PKC and displace phorbol esters, thus providing complex modulatory activity against a number of PKC isoforms, indicate bryostatins' potential for borad use in therapeutic applications outside of oncology, in treating any condition wherein modulation of PKC is beneficial.

Although the bryostatins have been known for some time, their low natural abundance, difficulties in isolation and severely limited availability through total synthesis have impeded efforts to elucidate their mode of action and to advance their clinical development. It is desired to provide new, simplified, and more readily accessible analogs of the natural bryostatins for use as PKC modulators.

Nomenclature

For simplicity of reference, the compounds of Formulae I-V are named and numbered herein as corresponding to the naturally occurring bryostatin macrocycle, described above with reference to Formula A.

For example, the analogues of the invention in which R²⁶ is hydrogen, such as those of Formula A-II and Formula A-III:

are also referred to as “C26 des-methyl”, notwithstanding that the structures corresponding to L (in Formula A.1) or the corresponding spacer domain, or even the recognition domain, contain fewer carbon atoms than native bryostatin such that the “C26” position would be assigned a lower number were these analogues to be named without reference to the native structure.

Further, as illustrated in the above representations of the Formulas, bonds represented with no substituents imply hydrogen substitution as valency permits. For example, Fragment A represents the same structure as Fragment B, differing in that all the hydrogens in the molecule are not expressly illustrated. For clarity, representations in the style of Fragment A may be used herein.

As used herein, the terms alkyl, alkenyl and alkynyl, refer to saturated and unsaturated monovalent moieties in accordance with their standard meanings, including straight-chain, branched-chain and cyclic moieties, optionally containing one or more intervening heteroatoms, such as oxygen, sulfur, and nitrogen in the chain or ring, respectively. Exemplary alkyl groups include methyl, ethyl, isopropyl, cyclopropyl, 2-butyl, cyclopentyl, pentyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, nonyl, decyl, and the like. Exemplary alkenyl groups include 2-pentenyl, 2,4-pentadienyl, 2-octenyl, 2,4,6-octatrienyl, CH₃—CH₂—CH₂—CH═CH—CH═CH—, cyclopentadienyl, and the like. Exemplary alkynyl groups include CH₃C≡CCH₂—, 4-pentyn-1-yl, and the like. Exemplary cyclic moieties include cyclopentyl, cyclohexyl, furanyl, pyranyl, tetrahydrofuranyl, 1,3-dioxanyl, 1,4-dioxanyl, pyrrolidyl, piperidyl, morpholino, and reduced forms of furanyl, imidazyl, pyranyl, pyridyl, and the like.

Lower alkyl, lower alkenyl, and lower alkynyl refer to alkyl, alkenyl, and alkynyl groups containing 1 to 4 carbon atoms.

The term aryl denotes a monocyclic or polycyclic aromatic ring or fused ring structure of carbon atoms with no heteroatoms in the ring(s). Examples are phenyl, naphthyl, anthracyl, and phenanthryl.

The term heteroatom is intended to have its customary meaning in the art and includes, but is not limited to oxygen, nitrogen and sulfur.

The term heteroaryl is used herein to denote a monocyclic or polycyclic aromatic ring or fused ring structure of carbon atoms with one or more non-carbon atoms, such as oxygen, nitrogen, and sulfur, in the ring or in one or more of the rings in fused ring structures. Examples are furanyl, pyranyl, thienyl, imidazyl, pyrrolyl, pyridyl, pyrazolyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, quinoxalyl, and quinazolinyl. Preferred examples are furanyl, imidazyl, pyranyl, pyrrolyl, and pyridyl.

The term heterocyclic is used herein to denote a monocyclic or polycyclic ring or fused ring structure which is saturated or partially unsaturated and containing one or more heteroatoms selected from the group consisting of O, S, and N. Nonlimiting examples are heterocycles such as thiofuranyl, pyranyl, dihydropyranyl, tetrahydropyranyl, pyrrolyl, tetrahydrothienyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolyl, oxazolidinyl, isoxazolidinyl, dioxazolyl, thiadiazolyl, tetrazolyl, triazolyl, thiatriazolyl, oxatriazolyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazoidinyl, dithiazolyl, diathiazolidinyl, tetrahydrofuryl, and benzofused derivatives thereof.

Aralkyl and heteroaralkyl refer to aryl and heteroaryl moieties, respectively, that are linked to a main structure by an intervening alkyl group, e.g., containing one or more methylene groups.

Alkoxy, alkenoxy, and alkynoxy refer to an alkyl, alkenyl, or alkynyl moiety, respectively, that is linked to a main structure by an intervening oxygen atom.

Alkylamino refers to alkyl moieties that are linked to a main structure by an intervening amino group.

Acyloxy refers to a moeity containing a carbonyl which is attached to a main structure by an intervening oxygen atom, such as for example; R′C(O)O—, ArC(O)O—, HetarylC(C)O—, and the like.

Acylamino refers to a moeity containing a carbonyl which is attached to a main structure by an intervening nitrogen atom, such as for example; R^(c)C(O)NR′—. ArC(O)NR′—, HetarylC(C)NR′—, and the like

It will be appreciated that the alkyl, alkenyl, alkynyl, alkoxy, alkylamino, acylamino, acyloxy, aryl, heteroaryl, aralkyl, and heteroaralkyl moieties utilized herein can be unsubstituted or substituted with one or more of the same or different substituents, which are typically selected from —X, —R′, ═O, —OR′, —SR′, ═S, —NR′R′, —NR′R′R′⁺, ═NR′, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O—, —S(O)₂OH, —S(O)₂R′, —C(O)R′, —C(O)X, —C(S)R′, —C(S)X, —C(O)OR′, —C(O)O—, —C(S)OR′, —C(O)SR′, —C(S)SR′, —C(O)NR′R′, —C(S)NR′R′ and —C(NR)NR′R′, where each X is independently a halogen (F, Cl, Br, or I, preferably F or Cl) and each R′ is independently hydrogen, alkyl, aryl, aralkyl, heteroaralkyl, heteroaryl, alkenyl, or alkynyl. In one embodiment, R′ is lower alkyl, lower alkenyl, or lower alkynyl. NR′R′ also includes moieties wherein the two R′ groups form a ring with the nitrogen atom, and may include other heteroatoms selected from the group consisting of O, S, and N, within the ring thus formed.

While practical size limits for the various substituent groups will be apparent to those skilled in the art, generally preferred are the alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and heteroaralkyl moieties containing up to about 40 carbon atoms, more preferably up to about 20 carbon atoms and most preferably up to about 10 carbon atoms (except as otherwise specifically noted, for example, with reference to the embodiment of the invention where a preferred R²⁰ substituent has about 7 to 20 carbon atoms).

As to any of the above groups that contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical, violate valency, and/or synthetically non-feasible. In addition, the compounds of this invention include all stereochemical isomers and mixtures thereof arising from the substitution of these compounds.

Except as otherwise specifically provided or clear from the context, the term “compounds” of the invention should be construed as including the “pharmaceutically acceptable salts” thereof (which expression has been eliminated in certain instances for the sake of brevity).

The term “pharmaceutically acceptable salt” refers to salts which retain the biological effectiveness and properties of the compounds of this invention and which are not biologically or otherwise undesirable. In some cases, the compounds of this invention are capable of forming acid and/or base salts, derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

Relatively lipophilic, as the term is used herein, describes a molecule, moiety, or region which is uncharged at neutral pH, and, taken alone, is only partially soluble in water. Relatively lipophilic moieties preferably have no more than one OH or NH bond for every five carbon atoms, even more preferably for every eight carbon atoms. Relatively lipophilic means that the molecule, moiety or region facilitates therapeutic use, helping to maintain a balance between lipophilicity (e.g., to permit cellular uptake) and hydrophilicity (e.g., to permit aqueous formulation).

Mammal is intended to have its conventional meaning. Examples include humans, mice, rats, guinea pigs, horses, dogs, cats, sheep, cows, etc.

The term treatment or treating means any treatment of a disease in a mammal, including:

-   -   preventing the disease, that is, causing the clinical symptoms         of the disease not to develop;     -   inhibiting the disease, that is, arresting the development of         clinical symptoms; and/or     -   relieving the disease, that is, causing the regression of         clinical symptoms.

The term effective amount means a dosage sufficient to provide treatment for the disease state being treated. This will vary depending on the patient, the disease and the treatment being effected.

Compounds of the Invention

Bryostatin is thought to act by modulating the activity and cellular localization of various Cl domain-containing proteins such as protein kinase C (PKC). In contrast to molecules that target the ATP binding site of PKC and function only as inhibitors, molecules that target the Cl domain can be designed to inhibit or activate enzyme activity. In addition, Cl domains are only present in a small subset of the large family of kinases, offering selectivity in function. The PKC family is divided into three subclasses: the conventional, novel, and atypical isozymes. Of these three, bryostatin binds only to the conventional and novel subclasses (eight isozymes in total). A long-standing goal in the area of Cl domain research is to design agents that can selectively regulate one or a subset of these eight isozymes. Compounds of the invention, with modifications to the “A” ring may provide selectivity between the isozymes as well as provide advantages in physicochemical behavior as therapeutic agents.

Various studies have demonstrated good affinity for bryostatins in which R^(A) is hydroxyl, acetyl, pivaloyl, or n-butanoate, and R^(B) is H, acetyl, n-butanoate, or 2,4-unsaturated octanoate, as measured by PKC binding assay. The double bond between C13 and C30 can be hydrogenated or epoxidized without significant loss of binding affinity. Hydrogenation of the C21-C34 alkene or acetylation of the C26 hydroxyl, on the other hand, can significantly reduce binding affinity. Inversion of the stereoconfiguration at C26 leads to modest loss of activity (approx. 30-fold) and the suggestion that the methyl group may limit rotation of bonds proximate to the methyl group and contribute to the apparent high binding affinity observed for the bryostatins. Elimination of the hydroxyl at C19 (with concomitant omission of the C20 R^(B) group) causes an approximately 100-fold to 200-fold decrease in binding.

The present invention provides new analogues of bryostatin that can be synthesized conveniently in high yields and which have useful biological activities. The compounds of the invention can be broadly described as having two main regions that are referred to herein as a “recognition domain” (or pharmacophoric region) and a relatively lipophilic “spacer domain” (or linker region). The recognition domain contains structural features that are analogous to those spanning C17 through C26 to C1, including the C ring formed in part by atoms C19 through C23, and the lactone linkage between C1 and C25 of the native bryostatin macrocycle. The spacer domain, on the other hand, joins the atoms corresponding to C1 through C17 of the native bryostatin macrocycle to substantially maintain the relative distance between the C1 and C17 atoms and the directionality of the C1C2 and C16C17 bonds, as illustrated by the arrows and distance “d” in Formula A.1 (in which the substituent groups are as defined with reference to Formula I).

In addition to its function of maintaining the recognition domain in an active conformation, the spacer domain (shown as “L” in Formula A.1 and sometimes also referred to as a linker region) provides a moiety that can be readily derivatized according to known synthetic techniques to generate analogues having improved in vivo stability and pharmacological properties (e.g., by modulating side effect profiles) while retaining biological activity.

It has been found in the present invention that the linker region of the bryostatin family can be varied significantly with retention of activity. Thus, a wide variety of linkers can be used while retaining significant anticancer and PKC-binding activities. Preferably, the compounds of the present invention include a linker moiety L, which is a linear, cyclic, or polycyclic linker moiety containing a continuous chain of from 6 to 14 chain atoms, one embodiment of which defines the shortest path from C25 via C1 to C17. Distance “d” should be about 2.5 to 5.0 angstroms, preferably about 3.5 to 4.5 angstroms and most preferably about 4.0 angstroms, such as about 3.92 angstroms (as experimentally determined, for example, by NMR spectroscopy). Thus, L may consist solely of a linear chain of atoms that links C17 via C1 to C25, or alternatively, may contain one or more ring structures which help link C17 via C1 to C25. Preferably, the linker region includes a lactone group (—C(═O)O—), or a lactam group (—C(═O)NH—), which is linked to C25 of the recognition region, by analogy to the C1 lactone moiety that is present in the naturally occurring bryostatins. In addition, it is preferred that the linker include a hydroxyl group analogous to the C3 hydroxyl found in naturally occurring bryostatins, to permit formation of an intramolecular hydrogen bond between the C3 hydroxyl of the linker and the C19 hydroxyl group of the recognition region (and optionally with the oxygen of the native B ring). In one preferred embodiment, the linker terminates with —CH(OH)CH₂C(═O)O—, for joining to C25 of the recognition region via an ester (or when cyclized, a lactone) linkage.

In one embodiment of the invention where R²⁶ is H, the compounds of the invention differ from known bryostatins and bryostatin analogues in that the present compounds contain a primary alcohol moiety at C26, i.e., the present analogues lack a methyl group corresponding to the C27 methyl that is ordinarily present in naturally occurring bryostatins. Surprisingly, while the C27 methyl moiety was previously believed to limit rotation of the C26 alcohol and contribute to PKC binding affinity, it has been found that this structural modification can significantly increase PKC binding and also increases efficacy against cancer cells.

In another aspect, the present invention provides bryostatins and bryostatin analogues in which R²⁰ is longer (e.g., having 9 to 20 or more carbon atoms) than the corresponding substituents at C20 in the native bryostatins (e.g., Bryostatin 3 having an 8-carbon atom moiety).

In one aspect of the invention, a compound of Formula I is provided

wherein X is O, S or NR′, R³ is H or OH; R⁷ is selected from the group consisting of optionally substituted lower alkyl, optionally substituted alkenyl, hydroxyl, amino, optionally substituted alkylamino, ═O, optionally substituted acylamino, OC(O)NR′R′, OC(O)OR′, OC(O)R′, and substituted acyloxy; p is 0, 1, 2, 3, or 4;

R²⁰ is H, OH, or -T-U—V—R′ where T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent;

R²¹ is ═CR^(a)R^(b) or R²¹ represents independent moieties R^(c) and R^(d) where R^(a) and R^(b) are independently H, CO₂R′, CONR^(c)R^(d) or R′; R^(c) and R^(d) are independently H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3;

R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl and optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof, with the proviso that the compound is not

wherein R″ is selected from the group consisting of CH₃, Ph, C₁₃H₂₇, C₇H₁₅, CH₂O(CH₂)₂O(CH₂)₂OCH₃, and

In some embodiments of the invention, a compound of Formula I is provided of the stereochemistry of Formula IA:

In some embodiments of the invention, a compound of Formula I is provided wherein R³ is OH. In other embodiments, a compound of Formula I is provided wherein R²⁶ is H as in Formula IB.

In some embodiments of the invention, exemplary compounds of Formula I have the structure of Formula IB. In some embodiments of the invention, exemplary compounds of Formula I have the structure of Formula IC. In some embodiments of the invention, exemplary compounds of Formula I have the structure of Formula ID. In some embodiments of the invention, exemplary compounds of Formula I have the structure of Formula IE.

The compounds of Formulas IB, IC, ID, and IE may have the stereochemical configuration of Formula IA as in the following formulas:

In another aspect of the invention, a compound of Formula II is provided

wherein R⁷ is selected from the group consisting of optionally substituted lower alkyl, optionally substituted alkenyl, hydroxyl, amino, optionally substituted alkylamino, ═O, optionally substituted acylamino, OC(O)NR′R′, OC(O)OR′, OC(O)R′, and substituted acyloxy; p is 0, 1, 2, 3, or 4;

R²⁰ is H, OH, or -T-U—V—R′ where T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent;

R²¹ is ═CR^(a)R^(b) or R²¹ represents independent moieties R^(c) and R^(d) where R^(a) and R^(b) are independently H, CO₂R′, CONR^(c)R^(d) or R′; R^(c) and R^(d) are independently H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3;

R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted aralkenyl, and optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof, with the proviso that the compound is not

wherein R″ is selected from the group consisting of CH₃, Ph, C₁₃H₂₇, C₇H₁₅, CH₂O(CH₂)₂O(CH₂)₂OCH₃, and

In some embodiments of the invention a compound of Formula II is provided of the stereochemistry of Formula IIA:

In some embodiments of the invention, a compound of Formula II is provided wherein R26 is H, as in Formula IIB. In some embodiments of the invention, a compound of Formula II with the structure of Formula IIC. In some embodiments of the invention, a compound of Formula with the structure of Formula IID. In some embodiments of the invention, a compound of Formula II with the structure of Formula IIE. All of the compounds of Formulas IIB, IIC, IID, and IIE may have the stereochemical configuration of Formula IIA.

Compounds of Formulas IIB, IIC, IID, and IIE may have the stereochemical configuration of Formula IIA, as in the following Formulas:

In a further aspect of the invention, a compound of Formula III is provided

wherein R³ is H or OH; R⁸ is selected from the group consisting of H, OH, R′, —(CH₂)O₂CR′, and —(CH₂)_(n)O₂C-haloalkyl; n is 0, 1, 2, 3, 4, or 5;

R⁹ is H or OH;

R²⁰ is H, OH, or -T-U—V—R′ where T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and

V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent;

R²¹ is ═CR^(a)R^(b) or R²¹ represents independent moieties R^(c) and R^(d) where R^(a) and R^(b) are independently H, CO₂R′, CONR^(c)R^(d) or R′; R^(c) and R^(d) are independently H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3;

R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted aralkenyl, and optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof, with the proviso that the compound is not

In some other embodiments of the invention, a compound of Formula III is provided having the stereochemical configuration of Formula IIIA:

In some embodiments a compound of Formula III is provided, having the structure of Formula IIIB. In some embodiments a compound of Formula III is provided, having the structure of Formula IIIC. In some embodiments a compound of Formula III is provided, having the structure of Formula IIID. In some embodiments a compound of Formula III is provided, having the structure of Formula IIIE. In some embodiments a compound of Formula III is provided, having the structure of Formula IIIF. In some embodiments a compound of Formula III is provided, having the structure of Formula IIIG. Any compound of Formulas IIIB, IIIC, IIID, IIIE, IIIF, and IIIG may have the stereochemical configuration of Formula IIIA.

The compounds of Formulas IIIB, IIIC, IIID, IIIE, IIIF, and IIIG may have the stereochemical configuration of Formula IIIA as in the following Formulas:

In another aspect of the invention, a compound of Formula IV is provided

wherein R³ is H or OH;

R⁷ is selected from the group consisting of optionally substituted lower alkyl, optionally substituted alkenyl, hydroxyl, amino, optionally substituted alkylamino, ═O, optionally substituted acylamino, OC(O)NR′R′, OC(O)OR′, OC(O)R′, and substituted acyloxy; p is 0, 1, 2, 3, or 4;

R²⁰ is H, OH, or -T-U—V—R′ where T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent;

R²¹ is H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3;

R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted aralkenyl, and optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof.

In some embodiments of the invention, a compound of Formula IV is provided having the stereochemical configuration of Formula IVA:

In some of the embodiments of the invention, compounds of Formula IV are provided wherein R³ is OH. In some of the embodiments of the invention, compounds of Formula IV are provided wherein R²⁶ is H.

In some embodiments, compounds of Formula IV have the following structures:

In some embodiments of the invention, the compounds of Formula IVB, IVC, IVD, and IVE have the stereochemical configurations as shown in the following Formulae:

Exemplary compounds of the invention include, but are not limited to

and their pharmaceutically acceptable salts thereof.

SYNTHESIS OF THE COMPOUNDS OF THE INVENTION Synthetic Reaction Parameters

The terms “solvent”, “inert organic solvent” or “inert solvent” mean a solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, pyridine and the like]. Unless specified to the contrary, the solvents used in the reactions of the present invention are inert organic solvents.

The terms “protecting group” or “blocking group” refer to any group which when bound to a functional group such as one or more hydroxyl, thiol, amino or carboxyl groups of the compounds (including intermediates thereof) prevents reactions from occurring at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, thiol, amino or carboxyl group. The particular removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl and any other group that can be introduced chemically onto a hydroxyl or like functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.

The term “q.s.” means adding a quantity sufficient to achieve a stated function, e.g., to bring a solution to the desired volume (i.e., 100%).

Unless specified to the contrary, the reactions described herein take place at atmospheric pressure within a temperature range from about 5° C. to 100° C. (preferably from 10° C. to 50° C.; most preferably at “room” or “ambient” temperature, e.g., 25° C.). Further, unless otherwise specified, the reaction times and conditions are intended to be approximate, e.g., taking place at about atmospheric pressure within a temperature range of about 5° C. to about 100° C. (preferably from about 10° C. to about 50° C.; most preferably about 25° C.) over a period of about 0.5 to about 10 hours (preferably about 1 hour). Parameters given in the Examples are intended to be specific, not approximate.

Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, distillation, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the general description and examples. However, other equivalent separation or isolation procedures can, of course, also be used.

Synthetic Methods

The compounds of the invention may be produced by any methods available in the art, including chemical and biological (e.g., recombinant and in vitro enzyme-catalyzed) methods. In one embodiment, the present invention provides a convergent synthesis in which subunits primarily corresponding to the recognition and spacer domains are separately prepared and then joined by esterification-macrotransacetalization. Additional syntheses of exemplary compounds of the invention are described below with reference to the Reaction Schemes. Other methods of synthesis may be used as known in the art. Some other methods which may be of use in synthesizing the compounds of the invention are found in D. A. Evans, et al., Angew. Chem. Int. Ed. 37:2354-2359 (1998); D. A. Evans, et al. J. Am. Chem. Soc. 121:7540-7552 (1999); S. Masamune, Pure Appl. Chem. 60:1587-1596 (1988); S. Masamune, Chimica 42:210-211 (1988); and P. D. Thiesen et al., J. Org. Chem. 53:2374-2378 (1988), and are incorporated herein by reference in their entirety. The stereochemical relationships illustrated for the various substituents should be taken to be optional. In some embodiments of the invention; the stereochemical relationships corresponding to the native bryostatins are provided in the compounds of the invention.

Reaction Scheme 1 illustrates synthesis of precursors for the recognition domain in compounds of the invention.

Reaction Scheme 2 illustrates the further synthesis of recognition domains for C26 des-methyl compounds of the invention.

Reaction Scheme 3 illustrates synthesis of the protected alcohol precursor to many of the C26 methyl analogues of the invention.

Reaction Scheme 4 illustrates the synthesis compounds which are suitable intermediates for “spacer” regions of bryostatin analogues.

Reaction Scheme 5A illustrates the synthesis compounds which are suitable intermediates for “spacer” regions of bryostatin analogues.

Reaction Scheme illustrates the synthesis compounds which are suitable intermediates for “spacer” regions of bryostatin analogues where R⁸ and/or R⁹ are H.

Reaction Scheme 5C illustrates the synthesis compounds which are suitable intermediates for “spacer” regions of bryostatin analogues where R⁶ is ═O with a variety of possible substituents at R⁸.

Reaction Scheme 6 illustrates synthetic routes which produce suitable intermediates for “spacer” regions of bryostatin analogues.

Reaction Scheme 7A illustrates a synthetic route for the compounds of Formula I and II.

Reaction Scheme 7B illustrates a synthetic route for the compounds of Formula III.

Reaction Scheme 8 illustrates synthesis of the Compounds of Formula V, particularly where R²⁶ is methyl, the C26 des-methyl analogues and compounds of Formula V where R¹² and R^(12a) are other than H being obtained by like synthesis.

Reaction Scheme 9 illustrates synthesis of the Compounds of Formula VI.

Reaction Schemes 10 and 11 illustrate the synthesis route that may be used to prepare compounds of the invention that are further derivatized at the C20 position, as discussed in Examples 4B, 4C and 4D.

Starting Materials. Conveniently, compounds of the invention can be prepared from starting materials that are commercially available or may be readily prepared by those skilled in the art using commonly employed synthetic methodology.

Reaction Scheme 1 illustrates a method for forming a synthon designated herein as 111 which is useful for providing the recognition domain in compounds of the invention, for example as detailed in Example 1. 6-(Tert-butyldimethylsilylhydroxy)-5-dimethylhexane-2,4-dione (101, Example 1B) is stirred with 2 equivalents of LDA (lithium diisopropylamine) in THF (tetrahydrofuran), followed by addition of 0.9 equivalents of 3R-p-methoxybenzyl-4R-benzyl-hydroxypentane-1-al (102, Example 1A) to afford diastereomeric aldol mixture 103 after suitable purification. To 103 is then added a catalytic amount of p-methylphenylsulfonic acid (p-TsOH) with stirring at room temperature followed by base quenching to produce pyranone condensation product 104 as a mixture of α and β-isomers at C23 (104a and 104b). The β-isomer (104a) is separated from the α-isomer and is reacted with NaBH₄ in the presence of CeCl₃.7H₂O, followed by quenching with aqueous brine to form an allylic alcohol (not shown) that can then be epoxidized with m-chloroperbenzoic acid (mCPBA) in 2:1 CH₂Cl₂:MeOH containing sodium bicarbonate as a buffer to yield a C19-methoxylated C20,C21 syn-diol 105. Selective benzoylation of the C21 equatorial alcohol with benzoyl chloride to afford C21 monobenzoate (not shown), followed by oxidation of the C20 hydroxyl with Dess-Martin periodinane (1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one) at room temperature affords the corresponding 20-keto-21-benzoate product 106. Treatment of 106 with SmI₂ (2 equiv) yields a ketone 107 selectively deoxygenated at C21. Next, ketone 107 is reacted with LDA and OHCCO₂CH₃ in THF at −78° C. to afford aldol mixture 108. After purification, 108 is reacted with methanesulfonylchloride in CH₂Cl₂ containing triethylamine, followed by reaction with DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in THF to effect an aldol condensation and elimination of water, to yield an α,β-unsaturated methyl ester (enone 109) with an E-stereoconfiguration. Treatment of enone 109 with NaBH₄ in the presence of CeCl₃.7H₂O produces exclusively the C20 axial alcohol 110. This product can then be esterified at C20, with octanoic acid for example, to yield the desired synthon 111.

It will be appreciated how the foregoing procedures can be exploited or modified to produce recognition region synthons having different substituents. For example, compounds where R²¹ contains a C35 ester group having a Z-configuration are produced during formation of intermediate 109 (Example 1C) and can be isolated by chromatography. Similarly, other ester groups can be introduced at C35 by replacing the OHCCO₂CH₃ reactant used to form 108 above with an appropriately substituted compound of the form OHCCO₂R′, in which R′ is other than methyl.

In addition, as detailed below, other substituents can be introduced in synthon 111 to generate substituent R²⁰ at C20 by substituting any of a variety of carboxylic acids for the octanoic acid reacted with axial alcohol 110 (as in the last step of Example 1C), including other saturated, unsaturated, aryl, and carboxylic acids. In synthesizing the compounds of the invention where R²⁰ has been varied, the substituent (e.g., a desired C20 ester substituent, carbonate, urea, thiourea, thiocarbamate or carbamate) can be introduced into a recognition region synthon prior to condensing the recognition region synthon with a linker synthon using the procedures described in Example 4 and via other synthetic routes well known to those skilled in the art. In Example 4A, the C20 octanoate substituent in synthon 111 can be replaced with an acetyl group by first protecting the base labile aldehyde group using trimethyl orthoformate to form the dimethyl acetal. The C20 octanoate ester can then be cleaved using a basic solution, such as K₂CO₃ in methanol, to afford the free C20 alcohol, followed by reaction with an activated form of acetic acid, such as acetic anhydride or acetyl chloride, to obtain the C20 acetate product. The product can then be deprotected at the C15 aldehyde, C19 oxygen, and C25 oxygen using benzoquinone compound DDQ (to remove the p-methoxybenzyl group and cleave the dimethyl acetal) followed by aqueous HF (demethylation at C19) to afford the corresponding C19 alcohol. This product can then be condensed with an appropriately substituted linker synthon to produce a desired bryostatin analogue, such as analogue 702.1, as detailed in Example 4A.

The protected alcohol precursor to many of the C26-desmethyl bryostatin analogues of the invention (the compounds of Formula A where R²⁶ is H) can be made as illustrated in Reaction Scheme 2. Di(benzyl ether) 111 can be hydrogenated over Pearlman's catalyst to produce the corresponding C25,C26 diol 201. Treatment of the diol with lead tetraacetate yields the corresponding C25 aldehyde (not shown), with the release of C26 and C27. Reaction of the aldehyde with Cp₂Ti(Cl)CH₂Al(CH₃)₂ (Tebbe's reagent) yields C25,C26 olefin 202. Alternatively, sodium periodate can be used in place of lead tetraacetate.

Treatment of olefin 202 with HF/pyridine is effective to remove the silyl protecting group, followed by treatment with Dess-Martin periodinane (supra) to oxidize the C₁₋₇ alcohol to an aldehyde group, affording aldehyde 203. The C25,C26 olefin of 203 can be converted to C25,C26 diol 204 by reaction with chiral dihydroxylating reagent (DHQD)₂AQN in the presence of K₃Fe(CN)₆, K₂CO₃ and K₂OsO₂(OH)₄ in t-butanol. Product 204 is obtained as a 2:1 (β:α) mixture of 25-hydroxy diastereomers. The α-diastereomer can be removed later in the synthesis. Treatment of 204 with triethylsilyl chloride yields protected diol 205, which can be employed in the synthesis of the compounds of Formula V.

Addition of backbone atoms corresponding to C15 and C16 of the bryostatin backbone to 205 can be accomplished in four steps. First, the C17 aldehyde is allylated with allyl diethylborane. The reaction is quenched with saturated sodium bicarbonate to yield the desired C17 alkyl adduct. The C17 hydroxyl group can then be acylated with acetic anhydride in the presence of triethylamine and 4-dimethylaminopyridine (DMAP), to afford a diastereomeric mixture of homoallylic C17 acetates. This product mixture can be oxidized using N-methylmorpholine N-oxide and osmium tetraoxide, followed by neutralization with sodium bicarbonate. After extraction, the residue is reacted with lead tetraacetate, followed by addition of DBU to cause elimination of the acetate group, yielding enal 206.

The C25 hydroxyl group of 206 can be unmasked in preparation for closure of the macrocycle as follows. First, enal 206 is treated with aqueous hydrofluoric acid to provide a crude diol intermediate in which the C19 methoxy group is converted to a free hydroxyl. Next, the diol product is reacted with tert-butyldimethylsilyl chloride (TBSCl) in the presence of imidazole to produce alcohol 207 containing a C25 hydroxyl group and C26 OTBS group as a 2:1 (β:α) mixture of C25 diastereomers. Silica gel chromatography can be used to resolve the diastereomers, affording the β diastereomer in 50-60% yield.

The protected alcohol precursor to many of the C26-methyl bryostatin analogues of the invention (Formula A where R²⁶ is methyl) can be made as illustrated in Reaction Scheme 3, via methods analogous to the preparations for 205 and 206. Deprotection and acylation of formula 111 may be accomplished by methods well known in the art.

Linker synthons for the compounds of Formulas I, II or IV can be prepared, for example, as illustrated with reference to Reaction Scheme 4, and later described in Examples 2A and 2B. These compounds contain two rings that are analogous to the A and B rings of bryostatin, but lack the naturally occurring substituents at C7, C8, C9, and C13. The presence of a heteroatom, such as an oxygen, sulfur or nitrogen atom (the lone electron pair of which is stabilized) in place of C14 does not adversely affect activity of the end product, but is required for transacetylization in the later synthetic steps. The compounds of formulae 406 and 408 differ in that 402 provides a protecting group precursor for a hydroxyl group attached to C3, whereas 406 does not provide for a hydroxyl at C3.

The linker synthons for the compounds of Formula III, which contain a B-ring-like structure but lack an A-ring, are prepared, for example, as illustrated with reference to Reaction Schemes 5A through 5C. Examples 2C and 2D describe methods for preparing synthons 504 and 508. In both examples, R⁸ is a tert butyl group attached to C9. However, with reference to the preparation of 505, the t-BuLi reactant can be replaced by R′Li to generate the corresponding linker synthons of 508 where R⁸ is R′. 504 additionally contains a TMS protecting group for synthesis of the compounds where R⁹ is a hydroxyl attached to C9, rather than hydrogen. Also, both synthons contain a TBS protecting group for the compounds where R³ is a hydroxyl group attached to C3. Example 2E describes the corresponding method for making synthon 507, which is unsubstituted at C9. Example 2G describes a method for preparing linker synthons in which C5 is provided as an ester carbonyl. In addition, the synthons in this Example contain an R⁶ substituent that is preferably a saturated or unsaturated substituent containing 1 to 20 carbon atoms and optionally (1) one or more oxygen atoms and (2) optionally one or more nitrogen atoms. In synthon 514 in Example 2G, R⁸ is —C(CH₃)₂CH₂C(═O)C₁₃H₂₇. However, other R⁶ substituents can be introduced by suitable modification of the procedure as will be evident to one of ordinary skill in the chemical arts.

Synthesis of a completely acyclic linker synthon 606 (where neither an A- nor a B-ring-like structure is present) is described with reference to Reaction Scheme 6 and in Example 2F.

As illustrated with reference to Reaction Scheme 7A, and further described in Example 3A, an alcohol such as 207, 303 or 304 is reacted with an acid such as 406 or 408 in a two step process to form the desired macrocyclic structure. After in situ conversion of the acid (408) to a mixed anhydride, the alcohol (207) is added to form ester 701. The ketal portion of 408 is then joined (in a process referred to as macrotransacetylization) to C15 of 701 by adding 70% HF/pyridine hydrofluoric acid to catalyze cleavage of the menthone ketal, cleavage of the TBS ethers at C3 and C26, and formation of a new ketal between the C15 aldehyde group and the linker diol moiety generated by release of the menthone (where X is oxygen), to afford desired analogue of Formulas I and II where X is a heteroatom and R²⁶ is H (starting with alcohol 207) or methyl (starting with 303 or 304), i.e., compound of formula 702. This last reaction is also effective to set the stereocenter at C15 to a thermodynamically preferred configuration. The analogous synthesis of compounds of Formula III, first forming the ether bond between C1 and C25, is illustrated with reference to Reaction Scheme 7B (where formula 703 corresponds to any of formulae 504, 507, 508 or 513) and further described in Example 3C.

As illustrated with reference to Reaction Scheme 8, and further described in Example 3B, the compounds of Formula V (such as formula 807) can be made from pharmacophoric synthon 801 and linker synthon 606 from Example 2F.

Reaction Scheme 9 illustrates synthesis of the compounds of Formula VI, such as compound 903,e.g., as further described in Example 3D, from synthon 111 and an activated dicarboxylic acid (succinic anhydride) to give formula 903.

Although the bryostatin analogues produced in Examples 3B, 3C and 3D all contain a C27 methyl group, analogous C26 desmethyl analogues can be readily synthesized using an appropriate C26 desmethyl synthon, such as C26 desmethyl synthon 207 described in Example IC and by other methods known in the art.

As illustrated with reference to Reaction Scheme 10, synthesis of a C20 heptanoate ester 43 is described in Example 4B, using a similar reaction scheme to that employed in Example 4A, except that heptenoic acid in the presence of triethylamine, DMAP, and Yamaguchi's agent is used in place of acetic anhydride. Yamaguchi's reagent is again employed in step f to activate the COOH group of formula 6, followed by removal of the TBS group in step g, hydrolysis of the menthone and transacetylization in step h, and saturation of the double bond upon removal of the benzyl group by hydrogenolysis in step i. Synthesis of a C20 myristate ester analogue 48 (14 carbon atom chain) is illustrated with reference to Reaction Scheme 11 and described in Example 4C. Reaction Scheme 11 and Example 4D describes synthesis of a bryostatin analogue containing an aryl ester group (benzoate) at C20, by suitable adaptation of the procedure in Example 1C for making compound 207. It will be appreciated how these procedures can be modified to introduce other C20 esters by substituting the starting materials necessary to produce the desired products. In particular, C26 des-methyl analogues can be made using an appropriate C26 des-methyl synthon, such as 207 noted above.

The lactam analogues of the invention (wherein the heteroatom attached to C25 is NH) are obtained by converting the C₂₋₅ hydroxyl group (e.g., of formula 207) to an amine under Mitsonobu conditions, after first protecting the aldehyde (and the C19 hydroxyl group in the corresponding compounds in Reaction Schemes 10 and 11) followed by formation of the macrocycle and de-protection under conditions analogous to those employed for the lactone analogues, as will be apparent to one skilled in the art. Lactam embodiments, can also be prepared by performing an aminohydroxylation reaction, instead of dihydroxylation with OsO₄. (employing protection/deprotection as described above). Chiral ligands for this reaction are known, and can be used to influence the stereochemical and/or regiochemical outcomes of the aminohydroxylation. This strategy can be employed on substrates in which the terminal alkene of the above scheme is further substituted, thereby providing access to compounds wherein R²⁶ is other than hydrogen. Such starting materials can be prepared by cleaving the olefin to the aldehyde (e.g., by OsO₄/periodate or ozonolysis) and performing a Wittig or other olefination reaction to obtain a desired secondary alkene.

The C26 des-methyl bryostatin homologues of the invention can be obtained by substituting homologous des-methyl starting materials for the starting materials. Other synthetic methodology will be apparent to those skilled in the art given the objective of providing such C26 des-methyl bryostatin homologues.

In certain embodiments, the fragment corresponding to the recognition domain (or C-ring portion) of bryostatin is prepared by a method that includes one or more of the steps illustrated with regard to Reaction Schemes 12 to 15, wherein:

R′, R²⁰ and R²⁶ are as defined above (for example, R²⁰ being —O₂CR′ or —O₂CNHR′);

R* represents, independently for each occurrence, H or a lower alkyl group such as methyl or ethyl;

q represents 0 or 1;

E represents an aldehyde, hydroxymethyl, carboxyl group, or a protected form thereof, such as CHO, CH₂OP, CH(OP)₂, or CO₂P;

P represents H or a protecting group, including protecting groups wherein two occurrences of P, taken together, form a ring having 5-7 members including the atoms through which they are connected (e.g., CH(OP)₂ may represent a cyclic acetal, e.g., with ethylene glycol or pinacol); and

G is absent or represents P.

With respect to the reactions illustrated in Reaction Schemes 13 to 15, the group identified as R′ is preferably hydrogen or lower alkyl (such as methyl or ethyl).

In certain embodiments wherein P is a protecting group for a hydroxyl, P represents a trialkylsilyl, dialkylarylsilyl, benzyl, substituted benzyl, benzhydryl, substituted benzhydryl, 5-dibenzosuberyl, triphenylmethyl, substituted triarylmethyl, naphthyldiphenylmethyl, 2- or 4-picolyl, 3-methyl-2-picolyl-N-oxide, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichloro-phthalamidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxy-phenyl)methyl, 9-(9-phenyl)xanthenyl, pivaloyl, adamantoyl, and 2,2,2-trichloroethylcarbonyl.

In certain embodiments wherein P is a protecting group for an acetal, P represents lower alkyl (such as methyl, ethyl, isopropyl, etc.) or, taken together with a second occurrence of P, represents a lower alkylene group (such as CH₂CH₂, CH₂CMe₂CH₂, etc.).

In certain embodiments wherein P is a protecting group for a carboxylic acid, P represents dialkylarylsilyl, benzyl, substituted benzyl, trichloroethyl, t-butyl, trimethylsilylethyl, lower alkyl, lower alkenyl (such as allyl), or other suitable protecting group.

In certain embodiments, E represents a carboxylic acid derivative that can be readily converted to an aldehyde, such as a thioester (reduction by triethylsilane in the presence of a transition-metal catalyst), CONMe(OMe) (reduction by a hydride reagent), an ester (reduction by diisobutylaluminum hydride (DIBAL-H)), etc. Other such groups and reaction conditions are well known to those of skill in the art.

The starting materials for Reaction Schemes 13 and 14 can be prepared, for example, as illustrated in Reaction Scheme 12.

In one embodiment, compound 12A can be converted to compound 12B by reaction with an acetone enolate or equivalent thereof, such as a lithium or magnesium anion of the N,N-dimethylhydrazone of acetone. Alternatively, the methyl ester of 12A can be reduced to an aldehyde (e.g., by reduction with DIBAL-H, or reduction by lithium aluminum hydride (LAH) followed by Swern oxidation), followed by aldol addition with an acetone enolate or equivalent and oxidation of the aldol product to the diketone. The dienolate of diketone 12B can then be reacted with aldehyde 12C to give aldol product 12F, which can be subsequently protected and/or reduced at the C²¹ ketone selectively (e.g., with tetramethylammonium triacetoxyborohydride, with an aldehyde in the presence of a Lewis acid via an intramolecular hydride transfer, etc.).

Alternatively, ester 12A can be converted to ketone 12E, e.g., by reduction to the aldehyde, reaction with methyllithium or methyl magnesium halide, and oxidation to the ketone. Aldehyde 12C can be extended to aldehyde 12D by aldol addition of an acetaldehyde equivalent (e.g., reaction with an allylsilane, allylborane, or allyltin reagent followed by ozonolysis or dihydroxylation/periodate cleavage, reaction with a silyl ketene acetal of an acetate ester in the presence of a Lewis acid catalyst such as TiCl₃(OiPr), TiCl₂(OiPr)₂, SnCl₄, or Sn(OTf)₂ followed by reduction to the aldehyde, reaction of an enolate of an acetate ester followed by reduction to the aldehyde, etc. For many of these reactions, chiral reagents or ligands are available that may be used to favor a desired diastereomer of the aldol product, while others may be sufficiently stereoselective without use of a chiral reagent. An enolate of ketone 12E can then be added to aldehyde 12D to arrive at aldol 12F by a different route.

Compounds wherein q is 0 can be readily converted to compounds wherein q is 1 as is described in detail above, e.g., by converting 12E to an aldehyde and performing a Horner-Emmons reaction with a phosphonoacetate reagent, performing a Peterson reaction with a trimethylsilylacetate reagent, or by other techniques known to those of skill in the art or described above.

Compounds useful in executing this strategy also include compounds represented by Formula 12G:

where, as valence and stability permit, q, E, P, G and R²⁶ are as defined above (with and without regard to the illustrated stereochemical relationships).

In Reaction Scheme 13 (below) Step a can be performed by treating the diketone compound 12F (produced as described in Reaction Scheme 12, where G at C₂₁ is absent) with acid, preferably under conditions that favor the removal of the water generated, such as distillation of the generated water (optionally as an azeotrope), addition of a dehydrating agent (such as molecular sieves, a carboxylic acid anhydride, or sodium or calcium sulfate), or other suitable means. If P on the C₂₃ hydroxyl represents a protecting group, the cyclization can be performed under conditions that selectively remove this protecting group, or this protecting group can be removed prior to the cyclization.

Step b can be performed by a series of reactions. For example, step b may begin with reduction of the ketone 13A with a hydride source, such as lithium or sodium borohydride, lithium aluminum hydride, etc. In certain embodiments, a reduction selective for the ketone over the unsaturation is performed, such as a Luche reduction (sodium borohydride in the presence of cerium (III) chloride heptahydrate). Oxidation of the alkene can then be performed with monoperoxyphthalate hexahydrate (MMPP), by epoxidation (e.g., with mCPBA, or dimethyldioxirane), or by dihydroxylation (e.g., with osmium tetroxide, optionally with an asymmetric ligand as is well known in the art). The product of the oxidation, if performed in the presence of water, will be the hemiketal, and if performed in the presence of an alcohol, will be the corresponding mixed ketal.

The present invention also encompasses the intermediate compounds useful in executing this strategy, including the compounds of Formulae 13A and 13B (with and without regard to the illustrated stereochemical relationships).

Compounds where R²⁰ is H can be prepared by a method including one or more steps of the following sequence:

Step a can be performed starting with a compound of Formula 12F (produced as described in Reaction Scheme 12, where G at C₂₁ is hydrogen) by an acid-catalyzed cyclization in a reaction mixture including an alcohol HOR*, preferably as a solvent or cosolvent, in order to form the mixed ketal. If P on the C23 hydroxyl represents a protecting group, the cyclization can be performed under conditions that selectively remove this protecting group (and possibly also any protecting group at C21), or one or both of these protecting groups can be removed prior to the cyclization.

Step b can be performed by oxidation of the C21 alcohol (e.g., by Swern oxidation, Dess-Martin periodinane, etc.), after removing any protecting group at this position, followed by reaction with a reagent such as R′O₂CCH₂SiMe₃ or R′O₂CCH₂PO₃Me₂. Optionally, a chiral phosophonoacetate (e.g., a phosphonate ester of BINOL, or other chiral diols or alcohols) or phosphonamidoacetate (e.g., a phosphonate derivative of ephedrine or another chiral aminoalcohol) may be employed in order to favor the desired stereochemical outcome of the enoate installation. Subsequent steps may be performed in analogy to well known procedures as discussed above.

In embodiments wherein q is 0, extension to embodiments wherein q is 1 can be readily accomplished by reaction of a compound wherein E is an aldehyde and q is 0 with a reagent such as Li—CH═CHOEt, ECH₂P(O)(OMe)₂, ECH₂SiMe₃, etc., wherein E represents an aldehyde or ester moiety. In certain preferred embodiments, a compound wherein E is aldehyde and q is 0 is converted to a compound wherein E is aldehyde and q is 1 by treatment with a preparation of a 2-alkoxyvinyllithium (such as 2-ethoxyvinyllithium) and a dialkylzinc (such as dimethylzinc), followed by treatment with acid, such as HCl, to convert the beta-hydroxyenol ether adduct to the unsaturated aldehyde, as is described in greater detail below. Other suitable reagents will be well known to those of skill in the art.

The present invention also encompasses the intermediate compounds useful in executing this strategy, including the compounds of Formulae 14A and 14B (with and without regard to the illustrated stereochemical relationships).

Alternatively, the C-ring fragment analog for compounds where R²⁶ is H can be prepared as illustrated in Reaction Scheme 15.

In this scheme, alcohol 15A can be oxidized to an aldehyde (e.g., by Swern or TPAP/NMO oxidation), followed by addition of a Grignard or lithium reagent derived from a 4-halo-1-butanol, such as 4-chloro- or 4-bromo-1-butanol. Both alcohols of 15B can then be oxidized (e.g., by Swern or TPAP/NMO oxidation), followed by selective addition of an allyl group to the aldehyde (e.g., by treatment with an allylstannane or allylsilane in the presence of a Lewis acid catalyst, optionally in the presence of a chiral ligand for the Lewis acid, such as BINOL, Ti(OPr)₄, and B(OMe)₃ together) to give ketoalcohol 15C. Cyclization and elaboration of this piece can be performed in analogy with the scheme presented above, for example, by cyclizing in the presence of an acid under dehydrating conditions, followed by oxidation of the enol ether with magnesium monoperoxyphthalate hexahydrate (MMPP), and oxidation of the resulting alcohol (e.g., by Swern or TPAP/NMO oxidation) to give ketone 15D. The exocyclic enoate can then be installed by an aldol condensation with methyl glyoxylate, e.g., by forming the lithium anion of ketone 15D with lithium diisopropylamide (LDA) under anhydrous conditions, or in an alcoholic solvent (such as methanol) in the presence of a base (such as sodium, potassium, or cesium carbonate). The terminal alkene can then be oxidized to the diol (e.g., by OsO₄ or certain hypervalent iodine reagents), optionally in the presence of a chiral ligand, as is well known in the art. Embodiments wherein q is 0 can be converted to embodiments wherein q is 1 as described in detail above.

The present invention also encompasses the intermediate compounds useful in executing this strategy, including the compounds of Formulae 15C, 15D, 15E and 15F (with and without regard to the illustrated stereochemical relationships).

In certain embodiments, a fragment analogous to the A and B rings of bryostatin can be prepared for incorporation into an analog of the present invention by a method including the steps illustrated in Reaction Scheme 16, where the substituents R*, R⁷ and P are as defined above:

Thus, a glutaric diester 16A can be condensed with a dienolate of an acetoacetate ester to provide diketoester 16B, followed by reduction of the two ketones to give diol 16C. One ketone can be reduced by a Noyori hydrogenation in the presence of a chiral catalyst to impart a first asymmetric center, and the second can be reduced stereoselectively using the newly formed stereocenter (tetramethylammonium triacetoxyborohydride for anti, Et₂BOMe and NaBH₄ for syn, for example). Acid-catalyzed lactonization followed by protection of the remaining alcohol to give lactone 16D provides a substrate for a second reaction with a dienolate of an acetoacetate ester to give ketoester 16E. Acid-catalyzed reduction of the hemiketal with triethylsilane, or dehydration of the hemiketal followed by hydrogenation, generates the tetrahydropyran of the A-ring analog. The remaining ketone can be reduced using a Noyori hydrogenation for a second time, or by hydride reduction in the presence of a chelating Lewis acid to take advantage of the tetrahydropyran stereochemistry, thereby producing alcohol 16F. Optionally, the terminal 1,3-diol can be converted to a ketal 16G in the presence of acid and a ketone, ketal, or enol ether.

The present invention also encompasses the intermediate compounds useful in executing this strategy, including the compounds of Formulae 16E, 16F and 16G (with and without regard to the illustrated stereochemical relationships). Compounds useful in executing this strategy also include compounds represented by Formulae 16H, 16I and 16J:

Reaction schemes 17 and 18 illustrate a synthetic route to prepare analogs of Formula II of the invention, as discussed in Example 14.

Conjugate addition of known homoallylic alcohol 17A (available in one step from 4-bromobenzaldehyde) to acrylonitrile to afford 17B (Reaction Scheme 17). Recrystallization of 17B from ethyl ether/pentane increased the enantiomeric purity from 92% to greater than 99% ee. The structure and absolute configuration of 17B were confirmed by X-ray crystallographic analysis.

Nitrile 17B was subsequently elaborated to β-ketoester 17C using a modified Blaise reaction. Ozonolytic cleavage of the terminal olefin generated an unstable aldehyde, which was immediately used in the next step without isolation. Addition of ethyl diazoacetate and SnCl₄ to the flask containing the aldehyde solution effected a Roskamp homologation that produced di-β-ketoester 17D in good yield. Double asymmetric reduction of di-β-ketoester 17D using Noyori's [(R)-BINAP]RuBr₂ catalyst cleanly produced di-β-3-hydroxyester 17E in high yield and with superb diastereoselectivity. HPLC analysis of the product mixture revealed that 17E was produced with 98% diastereomeric purity. This mixture could be enriched to greater than 99% isomeric purity by column chromatography. Selective reduction of the ethyl ester in the presence of the tert-butyl ester using lithium triethylborohydride afforded the corresponding triol. In an efficient one-step transformation, treatment of the triol with TBSOTf protected the three hydroxyl groups and cleaved the tert-butyl ester to reveal the Cl carboxylic acid. This concise route provided the completed spacer domain 17F in only seven steps and in 27% overall yield.

Spacer domain 17F was coupled to the recognition domain 18B using Yamaguchi's esterification protocol (Reaction Scheme 18). The macrocycle was then closed, and the silyl protecting groups were removed in a remarkably effective, one-step, mild, and diastereoselective macrotransacetalization providing completed analogue 18C. In this process, four silyl protecting groups are removed, and the macrocycle is closed. The newly created C15 stereocenter is formed under thermodynamic control, affording only the bryostatin-like stereochemistry. Hydrogenolysis of 18C using Pd/C provided the known desbromo analogue.

Reaction Scheme 19 illustrates a late stage cross coupling strategy to obtain diverse compounds of Formula III.

Diversification of analogue 18C can be achieved using cross-coupling chemistry. Suzuki coupling of 18C with phenylboronic acid using the Dave-Phos ligand produced new analogue 19B.1 (R=phenyl) in a single step. Analogue 19B.2 (R=—(CH₂)₃O(CH₂)₂OCH₂CH₃) was also produced in this manner. Further, vinylboronic acids could be smoothly coupled using the S-Phos ligand. Coupling of vinylboronic acids with analogue 18C cleanly produced new analogues 19B.3 (R=—C═C(CH₂)₅CH₃) and 19B.4 (R=—C═C(CH₂)₁₁CH₃₎.

Reaction Scheme 20 illustrates a process of synthesizing compounds of Formula IV:

Photoisomerization of analogue 20A at 254 nm in methylene chloride under nitrogen at room temperature for forty minutes cleanly provided analogue 20B.

Other Synthetic Transformations and Processes

A C19,C26 hydroxyl-protected, C26 des-methyl bryostatin recognition domain precursor and an optionally protected linker synthon are esterified, macrotransacetylated and de-protected to give the corresponding C26 des-methyl bryostatin analogue.

A bryostatin analogue precursor having the C26 hydroxyl substituted by a protecting group (particularly OBn) is reduced to give the corresponding compound of Formula I.

A pyran-4-ol of Formula 404 is converted to the corresponding pyran-2-yl-acetaldehyde of Formula 405 under reaction conditions including the presence of isobutylvinyl ether and Hg(II) diacetate. The reaction conditions further include carrying the crude vinylated pyran forward without delay, contacting it with anhydrous decane.

A diketone of Formula 12F is converted to the corresponding dihydropyranone of Formula 13A under reaction conditions including the presence of an acid, particularly where R²⁶ represents H or C₁ to C₆ alkyl. The reaction conditions can further include means for removing water.

A ketone of Formula 12F is converted to a tetrahydropyran of Formula 14A under reaction conditions including the presence of an acid and an alcohol (R*OH), particularly where R²⁶ represents H or C₁ to C₆ alkyl. The alcohol can be present as a solvent or cosolvent making up at least 20% of total solvent.

A ketone of Formula 15C is converted to the corresponding dihydropyranone of Formula 15D under reaction conditions including the presence of an acid. The reaction conditions can further include means for removing water.

A ketone of Formula 15D is converted to the corresponding ketoenoate of Formula 15E under reaction conditions including the presence of an alkyl glutarate ester.

An optionally protected lactone of Formula 16E is contacted with a dienolate of an ester of acetoacetate under conditions that provide the corresponding tetrahydropyran of Formula 16F.

A compound of Formula I-IV is contacted with a pharmaceutically acceptable acid to form the corresponding acid addition salt.

A pharmaceutically acceptable acid addition salt of Formula I-IV is contacted with a base to form the corresponding compound of Formula I-IV.

In some embodiments, a stereospecific synthesis for preparing a bryostatin analog is used, having a step selected from the group:

particularly where the starting material for Step 13a, 14a, 15c, 15d or 16d has one or more of the stereochemical configurations represented by formulae 12F, 12F, 15C, 15D or 16D, respectively, and especially where:

R⁷ is absent;

R²⁰ is R²⁰ is H, OH, —O₂C-lower alkyl or —O₂C-alkenyl;

R²⁶ is H or OH;

R′ is independently selected from: H and methyl;

R* is independently selected from: H and methyl;

q is 1;

E is OPMB, TBSO—CH₂— or —C(O)H; and/or

P is H, benzyl, OPMB or TBSO.

In some embodiments of the invention, a method of manufacture of an analogue of bryostatin of Formula I is provided comprising the steps of: converting an acid of a compound of Formula G-1 to a mixed anhydride; reacting said mixed anhydride of said compound of Formula G-1 with an alcohol of a compound of Formula G-2; transacetylizing a ketal of said compound of Formula G-1 with an aldehyde of said compound of Formula G-2; and removing protecting groups to yield said analogue of bryostatin of Formula II.

METHODS OF USE

Hosts, including mammals and particularly humans, suffering from any of the disorders described herein, including abnormal cell proliferation and PKC related disorders, can be treated by administering to the host an effective amount of a bryostatin analogue as described herein, or a pharmaceutically acceptable prodrug, solvate, ester, and/or salt thereof, optionally in the presence of a pharmaceutically acceptable carrier or diluent. The active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, transdermally, bronchially, pharyngolaryngeal, intranasally, topically, rectally, intracisternally, intravaginally, intraperitoneally, bucally or as an oral or nasal spray.

The active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to the host a therapeutically effective amount of compound to treat, for example, abnormal cell proliferation in vivo, without causing serious toxic effects in the host treated. It is to be understood that for any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.

The term pharmaceutically acceptable prodrug or prodrug, as used herein, represents those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of hosts, such as humans and mammals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. Prodrugs of the present invention may be rapidly transformed in vivo to a parent compound of formula (I), for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).

Bryostatin is thought to act by modulating the activity and cellular localization of various Cl domain-containing proteins such as protein kinase C (PKC). In contrast to molecules that target the ATP binding site of PKC and function only as inhibitors, molecules that target the Cl domain can be designed to inhibit or activate enzyme activity. Analogues of bryostatin offer the potential of select modulatory activity, offering a greater range of efficacy in therapeutic interventions of many types.

In addition, Cl domains are only present in a small subset of the large family of kinases, offering selectivity in function. The PKC family is divided into three subclasses: the conventional (α, βI, βII), novel (δ, ε, η, θ), and atypical isozymes. Of these three, bryostatin binds only to the conventional and novel subclasses (eight isozymes in total). A long-standing goal in the area of Cl domain research is to design agents that can selectively regulate one or a subset of these eight isozymes.

Protein kinase C mediates one arm of the signal transduction pathway proceeding through inositol phospholipid breakdown. This pathway is involved in the action of a broad range of cellular effectors, including growth factors and oncogenes, and indirectly affects other transduction pathways such as that of the cyclic AMP second messenger system. Therefore, modulating PKC activity using the compounds of the invention may offer approaches for pharmaceutical intervention in many therapeutic areas.

The compounds of the invention are can be used to treat PKC mediated diseases and disorders.

For example, the compounds of the invention find use as anticancer agents in mammalian subjects. Representative cancer conditions and cell types against which the compounds of the invention may be useful include melanoma, myeloma, chronic lymphocytic leukemia (CLL), AIDS-related lymphoma, non-Hodgkin's lymphoma, colorectal cancer, renal cancer, prostate cancer, cancers of the head, neck, stomach, esophagus, anus, or cervix, ovarian cancer, breast cancer, peritoneal cancer, and non-small cell lung cancer. The compounds appear to operate by a mechanism distinct from the mechanisms of other anticancer compounds, and thus can be used synergistically in combination with other anticancer drugs and therapies to treat cancers via a multimechanistic approach. The compounds of the invention exhibit potencies comparable to or better than previous bryostatins against many human cancer types.

In another example, the compounds of the invention can be used to strengthen the immune system of a mammalian subject, wherein a compound of the invention is administered to the subject in an amount effective to increase one or more components of the immune system for which modulation of PKC pathways is required, by inhibition or activation. For example, strengthening of the immune system can be evidenced by increased levels of T cells, antibody-producing cells, tumor necrosis factors, interleukins, interferons, and the like. Effective dosages may be comparable to those for anticancer uses, and can be optimized with the aid of various immune response assay protocols such as are known in the art (e.g., see U.S. Pat. No. 5,358,711, incorporated herein by reference). The compound can be administered prophylactically, e.g., for subjects who are about to undergo anticancer therapies, as well as therapeutically, e.g., for subjects suffering from microbial infection, burn victims, subjects with neuroendocrine disorders, diabetes, anemia, radiation treatment, or anticancer chemotherapy. The immunostimulatory activity of the compounds of the present invention is unusual among anticancer compounds and provides a dual benefit for anticancer applications. First, the immunostimulatory activity allows the compounds of the invention to be used in greater doses and for longer periods of time than would be possible for compounds of similar anticancer activity but lacking immunostimulatory activity. Second, the compounds of the present invention can offset the immunosuppressive effects of other drugs or treatment regimens when used in combination therapies.

In other examples, the disorders of abnormal cell proliferation include tumors and cancers, psoriasis, autoimmune disorders, disorders brought about by abnormal proliferation of mesangial cells (including human renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic micro-angiopathy syndromes, transplant rejection, and glomerulopathies), rheumatoid arthritis, Behcet's syndrome, acute respiratory distress syndrome (ARDS), ischemic heart disease, post-dialysis syndrome, leukemia, vasculitis, restenosis, chronic hypoxic pulmonary hypertension, lipid histiocytosis, acute and chronic nephropathies, atheroma, arterial restenosis, autoimmune diseases, and ocular diseases with retinal vessel proliferation (for example, diabetic retinopathy).

Other areas of application for which the compounds of the invention may be useful include disorders of associative memory storage. PKC signalling pathways have been observed to regulate points in the neurodegenerative pathophysiology of Alzheimer's disease (AD). Bryostatin-1 has been studied preclinically and has demonstrated to have cognitive restorative and antidepressant effects. This may be due to reduction of neuotoxic amyloid production and accumulation, activation of select PKC isoforms, induction of synthesis of proteins involved in long term memory, and restoration of stress induced inhibition of PKC activity. The compounds of the invention may have more selective activities in modulating specific PKC isoforms involved and decreased toxicity relative to the natural product, and a lack of tumor promoting ability (unlike other classes of PKC modulator compounds) thus providing utility as therapeutics to treat AD, depression and other cognitive and memory disorders.

The compounds of the invention may be useful in antiviral and antiproliferative therapies by activating PKC to render a diseased cell susceptible to killing by a second therapeutic agent, for example, ganclicovir and.or radiation, in the case of Epstein Barr Virus associated nasopharngeal carcinoma (NPC). This may also be a fruitful approach for combination therapies for other viral infections such as HIV and HSV.

Combination Therapy

Compounds of the present invention can be used in combination with other chemotherapeutic agents to treat cancer. In some embodiments, the combination may provide a synergistic therapeutic effect. The synergy is believed to arise from the effect of using two therapeutic agents which act through different mechanistic pathways. For example, Taxol and a bryostatin analog, when administered together, either in the same composition or separately, to a subject, may prevent neoplastic cells from mounting resistance as readily as is possible using only a single agent acting through a single mechanistic pathway or binding only at one site on the neoplastic cells. Synergy may be then provided in interactions between the compounds of the present invention and Taxol, for example, or with chemotherapeutic agents of other classes used to treat cancer and other proliferative and immune related disorders.

Compounds of the present invention can be used in combination or alternation with radiation and chemotherapy treatment, including induction chemotherapy, primary (neoadjuvant) chemotherapy, and both adjuvant radiation therapy and adjuvant chemotherapy. In addition, radiation and chemotherapy are frequently indicated as adjuvants to surgery in the treatment of cancer. The goal of radiation and chemotherapy in the adjuvant setting is to reduce the risk of recurrence and enhance disease-free survival when the primary tumor has been controlled. Chemotherapy is utilized as a treatment adjuvant for lung and breast cancer, frequently when the disease is metastatic. Adjuvant radiation therapy is indicated in several diseases including lung and breast cancers. Compounds of the present invention also are useful following surgery in the treatment of cancer in combination with radio- and/or chemotherapy. Compounds of the invention may be administered before, concomitantly, in the same composition, or after administering one or more additional active agents.

Active agents that can be used in combination with a protein kinase C modulator of the present invention include, but are not limited to, alkylating agents, antimetabolites, hormones and antagonists, protein kinase C modulators of other classes, microtubule stabilizers, radioisotopes, antibodies, as well as natural products, and combinations thereof. For example, a compound of the present invention can be administered with antibiotics, such as doxorubicin and other anthracycline analogs, nitrogen mustards, such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH) Other antineoplastic protocols include the use of a compound of the invention with another treatment modality, e.g., surgery or radiation, also referred to herein as “adjunct anti-neoplastic modalities.”

More specific examples of active agents useful for combination with compounds of the present invention, in both compositions and the methods of the present invention, include but are not limited to alkylating agents, such as nitrogen mustards (e.g., mechlorethanmine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil); nitrosureas, alkyl sulfonates, such as busulfan; triazines, such as dacarbazine (DTIC); antimetabolites; folic acid analogs, such as methotrexate and trimetrexate; pyrimidine analogs, such as 5-fluorouracil, fluorodeoxyuridine, gemcitabin, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, and 2,2′-difluorodeoxycytidine; purine analogs, such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2 chlorodeoxy-adenosine (cladribine, 2-CdA); natural products, including antimitotic drugs such as paclitaxel (Taxol®), vinca alkaloids (e.g., vinblastine (VLB), vincristine, and vinorelbine), Taxotere® (docetaxel), camptothecin, estramustine, estramustine phosphate, colchicine, bryostatin, combretastatin (e.g., combretastatin A-4 phosphate, combretastatin A-1 and combretastatin A-3, and their phosphates), dolastatins 10-15, podophyllotoxin, and epipodophylotoxins (e.g., etoposide and teniposide); antibiotics, such as actimomycin D, daunomycin (rubidomycin), doxorubicin (adriamycin), mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, dactinomycin, and tobramycin; enzymes, such as L-asparaginase; antibodies, such as HERCEPTIN® (Trastruzumab), RITUXAN® (Rituximab), PANOREX® (edrecolomab), ZEVALIN® (ibritumomab yiuxetan), MYLOTARGT® (gemtuzumab ozogamicin), and CAMPATH® (alemtuzumab); biological response modifiers, such as interferon-alpha, IL-2, G-CSF, and GM-CSF; differentiation agents; retinoic acid derivatives; radiosensitizers, such as metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, RSU 1069, EO9, RB 6145, SR4233, nicotinamide, 5-bromodeozyuridine, 5-iododeoxyuridine, and bromodeoxycytidine; platinum coordination complexes such as cisplatin and carboplatin; anthracenedione; mitoxantrone; substituted ureas, such as hydroxyurea; methylhydrazine derivatives, such as N-methylhydrazine (MIH) and procarbazine; adrenalcortical suppressants, such as mitotane (o,p′-DDD), aminoglutethimide; cytokines, such as interferon alpha, beta, and gamma and Interleukin 2 (IL-2); hormones and hormone antagonists, including adreno-corticosteroids/antagonists such as prednisone and its equivalents, dexamethasone, and aminoglutethimide; progestins, such as hydroxyprogesterone, caproate, medroxyprogesterone acetate, and megesterol acetate; estrogens, such as diethylstilbestrol, ethynyl estradiol, and their equivalents; antiestrogens, such as tamoxifen; androgens, such as testosterone propionate and fluoxymesterone, as well as their equivalents; antiandrogens, such as flutamide; gonadotropin-releasing hormone analogs, such as leuprolide; nonsteroidal antiandrogens, such as flutamide, and photosensitizers, such as hematoporphyrin and its derivatives, Photofrin®, benzoporphyrin and its derivatives, Npe6, tin etioporphyrin (SnET2), pheoboride-α, bacteriochlorophyll-α, naphthalocyanines, phthalocyanines, and zinc phthalocyanines.

In one particular embodiment, the compounds of the invention are administered in combination or alternation with a second agent selected from the group such as vincristine, cisplatin, ara-C, taxanes, edatrexate, L-buthionine sulfoxide, tiazofurin, gallium nitrate, doxorubicin, etoposide, podophyllotoxins, cyclophosphamide, camptothecins, dolastatin, and auristatin-PE, for example, and may also be used in combination with radiation therapy. In a preferred embodiment, the combination therapy entails co-administration of an agent selected from: ara-C, taxol, cisplatin and vincristine In a specific embodiment, the compound of the invention is administered in combination or alternation with taxol. In another embodiment, the compound is administered in combination or alternation with cisplatin. In yet another embodiment, the compound of the invention is administered in combination or alternation with vincristine. In a further embodiment, the compound of the invention is administered in combination or alternation with ara-C.

In some embodiments of the invention, a second agent having therapeutic activity via an immunosuppressive mechanism distinct from that of the compound of the invention is administered in combination or alternation with the compound of the invention. In other embodiments of the invention, a second agent having therapeutic activity via an immunosuppressive mechanism is administered in combination or in alternation with the compound of the invention. In some embodiments of the invention, said administration of the second agent is before, after, or concomitantly with the administration of the compound of the invention In some embodiments of the invention, administration of the compound of the invention is via an oral, intravenous, intraarterial, intramuscular, local, intraperitoneal, parenteral, transdermal, ocular, or intrathecal route. In some of the embodiments of the invention, the second agent is administered via the same route of administration as the compound of the invention. In some embodiments of the method of the invention, the administration of the second therapeutic agent is via a different route of administration from the compound of the invention.

PHARMACEUTICAL COMPOSITIONS

A therapeutically effective dose refers to that amount of the compound that results in achieving the desired effect. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio of LD₅₀ to ED₅₀. Compounds that exhibit high therapeutic indices (i.e., a toxic dose that is substantially higher than the effective dose) are preferred. The data obtained can be used in formulating a dosage range for use in humans. The dosage of such compounds preferably lies within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized.

The term host, as used herein, refers to a cell or organism that exhibits the properties associated with abnormal cell proliferation. The hosts are typically vertebrates, including both birds and mammals. It is preferred that the mammal, as a host or patient in the present disclosure, is from the family of Primates, Carnivora, Proboscidea, Perissodactyla, Artiodactyla, Rodentia, and Lagomorpha. It is even more preferable that the mammal vertebrate of the present invention be Canis familiaris (dog), Felis catus (cat), Elephas maximus (elephant), Equus caballus (horse), Sus domesticus (pig), Camelus dromedarious (camel), Cervus axis (deer), Giraffa camelopardalis (giraffe), Bos taurus (cattle/cows), Capra hircus (goat), Ovis aries (sheep), Mus musculus (mouse), Lepus brachyurus (rabbit), Mesocricetus auratus (hamster), Cavia porcellus (guinea pig), Meriones unguiculatus (gerbil), and Homo sapiens (human). Most preferably, the host or patient as used within the present invention is Homo sapiens (human). Birds suitable as hosts within the confines of the present invention include Gallus domesticus (chicken) and Meleagris gallopavo (turkey).

The term treating and its grammatical equivalents as used herein includes achieving a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

The compositions of the invention may be administered via oral, intravenous, intra-arterial, intramuscularly, local, intraperitoneally, parenteral, transdermal, ocular, or intrathecal routes.

Dosage forms for topical administration of a compound of this invention include powders, sprays, ointments and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants which can be required. Opthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.

Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active compound(s) which is effective to achieve the desired therapeutic response for a particular host, compositions and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the host being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

In the treatment or prevention of conditions which require abnormal cellular proliferation inhibition, an appropriate dosage level will generally be about 0.0000001 to 500 mg per kg host body weight per day which can be administered in single or multiple doses. In some embodiments, the dosage level is from about 0.0000001 mg/kg to about 250 mg/kg per day. In other embodiments, the dosage level is from about 0.0000005 mg/kg to about 100 mg/kg per day. A suitable dosage level may be from at least about 0.0000001 mg/kg to about 250 mg/kg per day, from at least about 0.00000005 mg/kg to about 100 mg/kg per day, or from at least about 0.000001 mg/kg to about 50 mg/kg per day. Within this range the dosage may be about 0.00000001 mg/kg to about 0.00005 mg/kg; 0.00005 mg/kg to about 0.05 mg/kg or about 0.0005 mg/kg to about 5.0 mg/kg per day. For some embodiments wherein administration is via oral administration, the compositions are provided in the form of tablets containing from about 0.0001 to about 1000 milligrams of the active ingredient, or at least about 0.0001, 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 5.0, 10.0, 15.0. 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, or about 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the host to be treated. The compositions may be administered on a regimen of 1 to 4 times per day, and in some embodiments, the compositions are administered once or twice per day. In some embodiments, the compositions are administered once a week. The course of treatment with a compound of the invention may be for about 1-about 30 days; about lto about 90 days, about 1 to about 120 days; or about 1 to about 180 days. The course of treatment with a compound of the invention, may be for about 1 to 45 days; from about 1 to about 28 days, from about 1 to about 21 days, from about 1 to about 14 days, or from about 1 to about 7 days.

It will be understood, however, that the specific dose level and frequency of dosage for any particular host may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the nature of the disorder of abnormal cell proliferation, the severity of the particular disorder, and the host undergoing therapy.

The compositions of the present invention can also be used as coatings on stents, including intraluminal stents, such as described in, for example, U.S. Pat. Nos. 6,544,544; 6,403,635; 6,273,913; 6,171,609; and 5,716,981.

The compound or a pharmaceutically acceptable ester, salt, solvate or prodrug can be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, including other drugs against abnormal cell proliferation.

Pharmaceutical compositions of the invention typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, antioxidants, and the like. In one embodiment, the composition may comprise from about 1% to about 75% by weight of one or more compounds of the invention, with the remainder consisting of suitable pharmaceutical excipients. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, for example. Appropriate excipients can be tailored to the particular composition and route of administration by methods well known in the art, e.g., (Gennaro, 1990). Additional guidance for formulations and methods of administration can be found in patent references concerning previously known bryostatins, such as U.S. Pat. Nos. 4,560,774 and 4,611,066 to Pettit et al., which are incorporated herein by reference.

Liquid compositions can be prepared by dissolving or dispersing compound (e.g., from about 0.5% to about 20% of final volume), and optional pharmaceutical adjuvants in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, ethanol and the like, to form a solution or suspension. Useful vehicles also include polyoxyethylene sorbitan fatty acid monoesters, such as TWEEN M 80, and polyethoxylated castor oils, such as Cremophor EL™ available from BASF (Wyandotte, Md.), as discussed in PCT Publ. No. WO 97/23208 (which is incorporated herein by reference), which can be diluted into conventional saline solutions for intravenous administration. Such liquid compositions are useful for intravenous administration. One such formulation is PET diluent which is a 60/30/10 v/v/v mixture of PEG 400, dehydrated ethanol, and TWEEN™-80. Liquid compositions may also be formulated as retention enemas.

The compounds of the invention may also be formulated as liposomes using liposome preparation methods known in the art. Preferably, the liposomes are formulated either as small unilamellar vesicles or as larger vesicles.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include for example the following components: a sterile diluent such as water for injection, saline solution, fixed oils, liposomes, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS).

Pharmaceutical compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants including immunostimulating factors (including immunostimulatory nucleic acid sequences, including those with CpG sequences), preservative agents, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Suspensions, in addition to the active compounds, may contain suspending agents, as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.

The active compounds can also be in micro- or nano-encapsulated form, if appropriate, with one or more excipients.

Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, dicalcium phosphate, and salicylic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, liposomes, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches, optionally mixed with degradable or nondegradable polymers. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

Compounds of the present invention may be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes may be used. The present compositions in liposome form may contain, in addition to the compounds of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the natural and synthetic phospholipids and phosphatidylcholines (lecithins) used separately or together. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y., (1976), p 33 et seq. and U.S. Pat. No. 4,522,811. For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound or its monophosphate, diphosphate, and/or triphosphate derivatives is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.

Controlled Release Formulations

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body or rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylacetic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

The field of biodegradable polymers has developed rapidly since the synthesis and biodegradability of polylactic acid was reported by Kulkarni et al. (“Polylactic acid for surgical implants,” Arch. Surg, 1966, 93, 839). Examples of other polymers which have been reported as useful as a matrix material for delivery devices include polyanhydrides, polyesters such as polyglycolides and polylactide-co-glycolides, polyamino acids such as polylysine, polymers and copolymers of polyethylene oxide, acrylic terminated polyethylene oxide, polyamides, polyurethanes, polyorthoesters, polyacrylonitriles, and polyphosphazenes. See, for example, U.S. Pat. Nos. 4,891,225 and 4,906,474 to Langer (polyanhydrides), 4,767,628 to Hutchinson (polylactide, polylactide-co-glycolide acid), and 4,530,840 to Tice, et al. (polylactide, polyglycolide, and copolymers). See also U.S. Pat. No. 5,626,863 to Hubbell, et al which describes photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled release carriers (hydrogels of polymerized and crosslinked macromers comprising hydrophilic oligomers having biodegradable monomeric or oligomeric extensions, which are end capped monomers or oligomers capable of polymerization and crosslinking); and PCT WO 97/05185 filed by Focal, Inc. directed to multiblock biodegradable hydrogels for use as controlled release agents for drug delivery and tissue treatment agents.

Degradable materials of biological origin are well known, for example, crosslinked gelatin. Hyaluronic acid has been crosslinked and used as a degradable swelling polymer for biomedical applications (U.S. Pat. No. 4,957,744 to Della Valle et. al.; “Surface modification of polymeric biomaterials for reduced thrombogenicity,” Polym. Mater. Sci. Eng., 1991, 62, 731-735]).

Many dispersion systems are currently in use as, or being explored for use as, carriers of substances, particularly biologically active compounds. Dispersion systems used for pharmaceutical and cosmetic formulations can be categorized as either suspensions or emulsions. Suspensions are defined as solid particles ranging in size from a few manometers up to hundreds of microns, dispersed in a liquid medium using suspending agents. Solid particles include microspheres, microcapsules, and nanospheres. Emulsions are defined as dispersions of one liquid in another, stabilized by an interfacial film of emulsifiers such as surfactants and lipids. Emulsion formulations include water in oil and oil in water emulsions, multiple emulsions, microemulsions, microdroplets, and liposomes. Microdroplets are unilamellar phospholipid vesicles that consist of a spherical lipid layer with an oil phase inside, as defined in U.S. Pat. Nos. 4,622,219 and 4,725,442 issued to Haynes. Liposomes are phospholipid vesicles prepared by mixing water-insoluble polar lipids with an aqueous solution. The unfavorable entropy caused by mixing the insoluble lipid in the water produces a highly ordered assembly of concentric closed membranes of phospholipid with entrapped aqueous solution.

U.S. Pat. No. 4,938,763 to Dunn, et al., discloses a method for forming an implant in situ by dissolving a nonreactive, water insoluble thermoplastic polymer in a biocompatible, water soluble solvent to form a liquid, placing the liquid within the body, and allowing the solvent to dissipate to produce a solid implant. The polymer solution can be placed in the body via syringe. The implant can assume the shape of its surrounding cavity. In an alternative embodiment, the implant is formed from reactive, liquid oligomeric polymers which contain no solvent and which cure in place to form solids, usually with the addition of a curing catalyst.

U.S. Pat. No. 5,718,921 discloses microspheres comprising polymer and drug dispersed there within. U.S. Pat. No. 5,629,009 discloses a delivery system for the controlled release of bioactive factors. U.S. Pat. No. 5,578,325 discloses nanoparticles and microparticles of non-linear hydrophilic hydrophobic multiblock copolymers. U.S. Pat. No. 5,545,409 discloses a delivery system for the controlled release of bioactive factors. U.S. Pat. No. 5,494,682 discloses ionically cross-linked polymeric microcapsules.

U.S. Pat. No. 5,728,402 to Andrx Pharmaceuticals, Inc. describes a controlled release formulation that includes an internal phase which comprises the active drug, its salt, ester or prodrug, in admixture with a hydrogel forming agent, and an external phase which comprises a coating which resists dissolution in the stomach. U.S. Pat. Nos. 5,736,159 and 5,558,879 to Andrx Pharmaceuticals, Inc. discloses a controlled release formulation for drugs with little water solubility in which a passageway is formed in situ. U.S. Pat. No. 5,567,441 to Andrx Pharmaceuticals, Inc. discloses a once-a-day controlled release formulation. U.S. Pat. No. 5,508,040 discloses a multiparticulate pulsatile drug delivery system. U.S. Pat. No. 5,472,708 discloses a pulsatile particle based drug delivery system. U.S. Pat. No. 5,458,888 describes a controlled release tablet formulation which can be made using a blend having an internal drug containing phase and an external phase which comprises a polyethylene glycol polymer which has a weight average molecular weight of from 3,000 to 10,000. U.S. Pat. No. 5,419,917 discloses methods for the modification of the rate of release of a drug to form a hydrogel which is based on the use of an effective amount of a pharmaceutically acceptable ionizable compound that is capable of providing a substantially zero-order release rate of drug from the hydrogel. U.S. Pat. No. 5,458,888 discloses a controlled release tablet formulation.

U.S. Pat. No. 5,641,745 to Elan Corporation, plc discloses a controlled release pharmaceutical formulation which comprises the active drug in a biodegradable polymer to form microspheres or nanospheres. The biodegradable polymer is suitably poly-D,L-lactide or a blend of poly-D,L-lactide and poly-D,L-lactide-co-glycolide. U.S. Pat. No. 5,616,345 to Elan Corporation plc describes a controlled absorption formulation for once a day administration that includes the active compound in association with an organic acid, and a multi-layer membrane surrounding the core and containing a major proportion of a pharmaceutically acceptable film-forming, water insoluble synthetic polymer and a minor proportion of a pharmaceutically acceptable film-forming water soluble synthetic polymer. U.S. Pat. No. 5,641,515 discloses a controlled release formulation based on biodegradable nanoparticles. U.S. Pat. No. 5,637,320 discloses a controlled absorption formulation for once a day administration. U.S. Pat. Nos. 5,580,580 and 5,540,938 are directed to formulations and their use in the treatment of neurological diseases. U.S. Pat. No. 5,533,995 is directed to a passive transdermal device with controlled drug delivery. U.S. Pat. No. 5,505,962 describes a controlled release pharmaceutical formulation.

In one embodiment of the invention, stents are provided which comprise a generally tubular structure, which contains or is coated, filled or interspersed with compounds of the present invention, optionally with one or more other anti-angiogenic compounds and/or compositions. Methods are also provided for expanding the lumen of a body passageway, comprising inserting the stent into the passageway, such that the passageway is expanded.

The stents can be provided for eliminating biliary obstructions by inserting a biliary stent into a biliary passageway; for eliminating urethral obstructions by inserting a urethral stent into a urethra; for eliminating esophageal obstructions by inserting an esophageal stent into an esophagus; and for eliminating trachealibronchial obstructions by inserting a tracheal/bronchial stent into the trachea or bronchi.

In one embodiment of the present invention, the compound of the present invention is delivered to the site of arterial injury via a stent. In one approach, the therapeutic agent is incorporated into a polymer material which is then coated on or delivered onto or incorporated into at least a portion of the stent structure. To improve the clinical performance of stents, a therapeutic agent can be applied as a coating to the stent, attached to a covering or membrane, embedded on the surface material via ion bombardment or dripped onto the stent or to holes or reservoirs in a part of the stent that act as reservoirs. Therefore, in one embodiment of the present invention, the compounds are applied, attached, dripped and/or embedded to the stent by known methods.

The stents can be designed from a single piece of metal, such as from wire coil or thin walled metal cylinders, or from multiple pieces of metal. In a separate embodiment, the stents are designed from biodegradable materials such as polymers or organic fabrics. In one embodiment, the surface of the stent is solid. The stent is generally thin walled and can include a number of struts and optionally a number of hinges between the struts that are capable of focusing stresses.

In one embodiment, the stent structure includes a plurality of holes or, in a separate embodiment, a plurality of recesses which can act as reservoirs and may be loaded with the drug. The stent can be designed with particular sites that can incorporate the drug, or multiple drugs, optionally with a biodegradable or non-biodegradable matrix. The sites can be holes, such as laser drilled holes, or recesses in the stent structure that may be filled with the drug or may be partially filled with the drug. In one embodiment, a portion of the holes are filled with other therapeutic agents, or with materials that regulate the release of the drug or drugs. One advantage of this system is that the properties of the coating can be optimized for achieving superior biocompatibility and adhesion properties, without the addition requirement of being able to load and release the drug. The size, shape, position, and number of reservoirs can be used to control the amount of drug, and therefore the dose delivered.

In another embodiment, the surface of the stent can be coated with one or more compositions containing the compound of the invention. In one embodiment, a coating or membrane of biocompatible material could be applied over the reservoirs which would control the diffusion of the drug from the reservoirs to the artery wall. The coating may also be a sheath covering the surface of the stent. The coating may also be interspersed on the surface of the stent. Coatings or fillings are generally accomplished by dipping, spraying or printing the drug on or into the stent, for example through ink jet type techniques.

The compounds of the present invention are optionally applied in non-degradable microparticulates or nanoparticulates or biodegradable microparticulates or nanoparticulates. In one embodiment, the microparticles or nanoparticles are formed of a polymer containing matrix that biodegrades by random, nonenzymatic, hydrolytic scissioning, such as a structure formed from a mixture of thermoplastic polyesters (e.g., polylactide or polyglycolide) or a copolymer of lactide and glycolide components. The lactide/glycolide structure has the added advantage that biodegradation thereof forms lactic acid and glycolic acid, both normal metabolic products of mammals.

The present invention also provides therapeutic methods and therapeutic dosage forms involving administration of the compounds of the invention in combination with an inhibitor of vascular smooth muscle cell contraction to a vascular lumen, allowing the normal hydrostatic pressure to dilate the vascular lumen. Such contraction inhibition may be achieved by actin inhibition, which is preferably achievable and sustainable at a lower dose level than that necessary to inhibit protein synthesis. Consequently, the vascular smooth muscle cells synthesize protein required to repair minor cell trauma and secrete interstitial matrix, thereby facilitating the fixation of the vascular lumen in a dilated state near its maximal systolic diameter. This phenomenon constitutes a biological stenting effect that diminishes or prevents the undesirable recoil mechanism that occurs in up to 25% of the angioplasty procedures classified as successful based on an initial post-procedural angiogram. Cytochalasins (which inhibit the polymerization of G- to F-actin which, in turn, inhibits the migration and contraction of vascular smooth muscle cells) are the preferred therapeutic agents for use in this embodiment of the present invention. Free therapeutic agent protocols of this type effect a reduction, a delay, or an elimination of stenosis after angioplasty or other vascular surgical procedures. Preferably, free therapeutic agent is administered directly or substantially directly to vascular smooth muscle tissue. Such administration is preferably effected by an infusion catheter, to achieve a 10⁻³ M to 10⁻¹² M concentration of said therapeutic agent at the site of administration in a blood vessel.

The compounds of the present invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. By “pharmaceutically acceptable salt” is meant those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well-known in the art. For example, P. H. Stahl, et al. describe pharmaceutically acceptable salts in detail in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” (Wiley VCH, Zürich, Switzerland: 2002). The salts can be prepared in situ during the final isolation and purification of the compounds of the present invention or separately by reacting a free base function with a suitable organic acid. Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which can be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid.

Basic addition salts can be prepared in situ during the final isolation and purification of compounds of this invention by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like. Preferred salts of the compounds of the present invention include phosphate, tris and acetate.

IN-VITRO AND IN-VIVO TESTING

In practicing various aspects of the present invention, compounds in accordance with the invention can be tested for a biological activity of interest using any assay protocol that is predictive of activity in vivo. For example, a variety of convenient assay protocols are available that are generally predictive of anticancer activity in vivo.

In one approach, anticancer activity of compounds of the invention can be assessed using the protein kinase C assay detailed in Example 5. In this assay, K_(i) values are determined for analogues based on competition with radiolabeled phorbol 12,13-dibutyrate for binding to a mixture of PKC isoenzymes. PKC enzymes are implicated in a variety of cellular responses which may be involved in the activity of the bryostatins.

Example 6 describes another protein kinase C assay which can be used to assess the binding affinities of compounds of the invention for binding to the C IB domain of PKCδ. Although all PKC isozymes are upregulated immediately after administration of bryostatin or tumor promoting phorbol esters followed by an extended down-regulation period, PKCδ appears to be protected against down regulation by bryostatin 1. Overexpression of PKCδ inhibits tumor cell growth and induces cellular apoptosis, whereas depleting cells of PKCδ can cause tumor promotion. Accordingly, this assay provides useful binding data for assessing potential anticancer activity.

Another useful method for assessing anticancer activities of compounds of the invention involves the multiple-human cancer cell line screening assays run by the National Cancer Institute. This screening panel, which involves approximately 60 different human cancer cell lines, is a useful indicator of in vivo antitumor activity for a broad variety of tumor types, such as leukemia, non-small cell lung, colon, central nervous system (CNS), melanoma, ovarian, renal, prostate, and breast cancers. Antitumor activities can be expressed in terms of ED₅₀ (or GI₅₀), where ED₅₀ is the molar concentration of compound effective to reduce cell growth by 50%. Compounds with lower ED₅₀ values tend to have greater anticancer activities than compounds with higher ED₅₀ values. Example 7 describes a P388 murine lymphocytic leukemia cell assay which measures the ability of compounds of the invention to inhibit cellular growth.

Upon the confirmation of a compounds potential activity in the above in vitro assays, further evaluation is typically conducted in vivo in laboratory animals, for example, measuring reduction of lung nodule metastases in mice with B16 melanoma. The efficacy of drug combination chemotherapy can be evaluated, for example, using the human B-CLL xenograft model in mice. Ultimately, the safety and efficacy of compounds of the invention are evaluated in human clinical trials.

Experiments conducted in support of the present invention demonstrate that compounds of the present invention exhibit high potencies in several anticancer assays, as summarized in the Examples.

EXAMPLES General Techniques

Unless noted otherwise, materials were obtained from commercially available sources and used without further purification. Tetrahydrofuran (THF) and diethyl ether (Et₂O) were distilled from sodium benzophenone ketyl under a nitrogen atmosphere. Benzene, dichloromethane (CH₂Cl₂), acetonitrile (CH₃CN), triethylamine (Et₃N) and pyridine were distilled from calcium hydride under a nitrogen atmosphere. Chloroform (CHCl₃), carbon tetrachloride (CCl₄) and deuterated NMR solvents were dried over 1/16″ bead 4 Å molecular sieves.

All operations involving moisture-sensitive materials were conducted in oven- and/or flame-dried glassware under an atmosphere of anhydrous nitrogen. Hygroscopic solvents and liquid reagents were transferred using dry Gastight™ syringes or cannulating needles. In cases where rigorous exclusion of dissolved oxygen was required, solvents were degassed via consecutive freeze, pump, thaw cycles or inert gas purge.

Nuclear magnetic resonance (NMR) spectra were recorded on either a Varian UNITY INOVA-500, XL-400 or Gemini-300 magnetic resonance spectrometer. ¹H chemical shifts are given in parts per million (6) downfield from tetramethylsilane (TMS) using the residual solvent signal (CHCl₃=δ 7.27, benzene=δ 7.15, acetone=δ 2.04) as internal standard. Proton (1H) NMR information is tabulated in the following format: number of protons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; sept, septet, m, multiplet), coupling constant(s) (J) in hertz and, in cases where mixtures are present, assignment as the major or minor isomer, if possible. The prefix app is occasionally applied in cases where the true signal multiplicity was unresolved and br indicates the signal in question was broadened. Proton decoupled ¹³C NMR spectra are reported in ppm (δ) relative to residual CHCl₃ (δ 77.25) unless noted otherwise.

Infrared spectra were recorded on a Perkin-Elmer 1600 series FTIR using samples prepared as thin films between salt plates. High-resolution mass spectra (HRMS) were recorded at the NIH regional mass spectrometry facility at the University of California, San Francisco. Fast Atom Bombardment (FAB) high-resolution mass spectra were recorded at the University of California, Riverside. Combustion analyses were performed by Desert Analytics, Tucson, Ariz., 85719 and optical rotations were measured on a Jasco DIP-1000 digital polarimeter.

Flash chromatography was performed using E. Merck silica gel 60 (240-400 mesh) according to the protocol of Still et al. (1978). Thin layer chromatography was performed using precoated plates purchased from E. Merck (silica gel 60 PF254, 0.25 mm) that were visualized using either a p-anisaldehyde or Ce(IV) stain.

For binding and cell-inhibition studies, dilutions of bryostatin and bryostatin analogues were performed in glass rather than plastic, to avoid problems associated with adsorption to plastic.

Example 1 Exemplary Precursors 1A. Protected Diol Aldehyde 102

Benzyl bromide (7.0 mL, 57.7 mmol) and freshly prepared Ag₂O (11.0 g, 48.1 mmol) were added successively to an Et₂O (150 mL) solution of R-(+)-methyl lactate (5.0 g, 48.1 mmol) at rt (room temperature). The resulting suspension was brought to reflux and stirred for 2 h. The reaction was cooled to rt, filtered through a pad of Celite™ and concentrated in vacuo. Chromatography on silica gel (10% EtOAc/hexanes) afforded 7.5 g (80%) of benzyl ether A1 as a colorless oil:

A1: R_(f) (15% EtOAc/hexanes)=0.66; IR 2988, 2952, 2874, 1750, 1497, 1454, 1372, 1275, 1207, 1143, 1066, 1025, 739, 698 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 1.44 (3H, d, J=6.8 Hz, C27), 3.75 (3H, S, CH₃O), 4.07 (1H, q, J=6.8 Hz, C26), 4.45 (1H, d, J=11.7 Hz, CH₂Ph), 4.69 (1H, d, J=11.7 Hz, CH₂Ph), 7.28-7.37 (5H, m, Ph); ¹³C NMR (75 M Hz, CDCl₃) δ 18.6, 51.8, 71.9, 73.9, 127.7, 127.8, 128.3, 137.4, 173.6; HRMS Calcd for C₁₁H₁₄O₃ (M⁺): 194.0943. Found: 194.0942.

To a solution of methyl ester A1 (6.3 g, 32.3 mmol) in Et₂O (150 mL) was added DIBAL-H (1.0M in hexanes, 38.75 mL) dropwise at −78° C. via cannulating needle. After 5 min at −78° C., the reaction was quenched with H₂O and gradually warmed to rt. The resultant thick emulsion was filtered through a pad of Celite™ and sand, rinsing thoroughly with Et₂O and EtOAc. The organic phase was washed with NaHCO₃ (2×), dried over MgSO₄ and concentrated in vacuo to afford crude aldehyde A2 (not shown) as a light yellow liquid.

To a solution of SnCl₄ (1.0M in CH₂Cl₂, 32.3 mmol) in CH₂Cl₂ (120 mL) was slowly added a CH₂Cl₂ solution of aldehyde A2 at −78° C. The mixture was stirred for an additional 10 min before allyltrimethylsilane (5.65 mL, 35.53 mmol) was added via syringe. The ensuing white suspension was kept at −78° C. for 10 min, quenched by addition of H₂O and allowed to warm to rt. The aqueous layer was extracted with Et₂O and the combined organics were dried over MgSO₄ and concentrated in vacuo. Chromatography on silica gel (10% EtOAc/hexanes) afforded 4.87 g (76%) of desired diastereomer A3 plus 300 mg (5%) of a putative mixture.

A3: R_(f) (15% EtOAc/hexanes)=0.44; IR (film) 3454, 3066, 3030, 2976, 2871, 1641, 1497, 1554, 1375, 1071, 1028, 992, 914, 737, 698 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 1.20 (3H, d, J=6.2 Hz, C27), 2.22 (1H, m, C24), 2.35 (1H, m, C24), 2.56 (1H, br s, OH), 3.45 (1H, m, C25), 4.25 (1H, m, C26), 4.44 (1H, d, J=11.5 Hz, CH₂Ph), 4.66 (1H, d, J=11.5 Hz, CH₂Ph), 5.10 (2H, m, CH₂═CH), 5.87 (1H, m, CH₂═CH), 7.28-7.35 (5H, m, Ph); ¹³C-NMR (75 M Hz, CDCl₃) δ 15.3, 37.4, 70.9, 74.1, 77.3, 117.03, 127.6, 127.7, 128.3, 134.7, 138.2; HRMS Calcd for C₁₃H₁₈O₂ (M⁺): 206.1307. Found: 206.1313.

To a suspension of potassium tert-butoxide (4.0 g, 35.7 mmol) in 120 mL anhydrous THF was added a solution of alcohol A3 (in 30 mL of THF) slowly over 15 min at 0° C. When complete, the mixture was stirred at rt for 45 min and then warmed to 60° C. for an additional 30 min. p-Methoxybenzylchloride (3.56 mL, 26 mmol) was added and the mixture was stirred at 60° C. for 4 h. The reaction was cooled to rt and quenched with sat. NH₄Cl. The aqueous layer was extracted with Et₂O (3×) and the combined organics were dried over MgSO₄ and concentrated in vacuo. The crude product was purified by flash chromatography (5% EtOAc/hexanes) to provide 6.80 g (88%) of differentially protected diol A4 as a colorless oil.

A4: R_(f) (15% EtOAc/hexanes)=0.59; IR (film) 3065, 2935, 2868, 1641, 1613, 1586, 1514, 1464, 1380, 1302, 1248, 1094, 1037, 913, 821, 737 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 1.17 (3H, d, J=6.3 Hz, C27), 2.27 (1H, m, C24), 2.40 (1H, m, C24), 3.44 (1H, ddd, J=7.7, 4.7, 4.6 Hz, C25), 3.62 (1H, dq, J=7.7, 6.3 Hz, C26), 3.78 (3H, s, CH₃O), 4.51 (1H, d, J=11.9 Hz, CH₂Ph), 4.52 (2H, s, CH₂Ph), 4.60 (1H, d, J=11.9 Hz, CH₂Ph), 5.04 (2H, m, CH₂═CH), 5.84 (1H, m, CH═CH₂), 6.83 (2H, d, J=8.7 Hz, Ar), 7.23-7.33 (7H, m, Ar); ¹³C-NMR (75 M Hz, CDCl₃) δ 15.0, 34.4, 55.1, 71.2, 72.1, 75.7, 80.9, 113.6, 116.6, 127.4, 127.6, 127.6, 128.3, 129.4, 130.9, 135.6, 138.9, 159.2; HRMS Calcd for C₂₁H₂₆O₃ (M⁺): 326.1882. Found: 326.1876; Anal. Calcd for C₂₁H₂₆O₃: C, 77.27; H, 8.03. Found: C, 77.22; H, 8.16; [α]_(D) ²⁰=−8.9° (c 1.43, CH₂Cl₂).

Formula 102

A4 (3.0 g, 9.2 mmol) was dissolved in 90 mL CH₂Cl_(2/22.5) mL MeOH and cooled to −78° C. Ozone was bubbled through the solution which was carefully monitored for the disappearance of starting material by TLC (thin layer chromatography). When the consumption of A4 was judged complete, the system was immediately purged with N₂ for 20 min and treated with solid thiourea (840 mg, 11.04 mmol). The reaction was warmed to rt slowly over 5 hours and stirred at rt for 6 hours. The solvents were removed in vacuo, and the crude mixture was purified by flash chromatography (20% EtOAc/hexanes) to afford 2.40 g (80%) of aldehyde 102 as a colorless oil.

102: R_(f) (15% EtOAc/hexanes)=0.31; IR (film) 2868, 2729, 1723, 1612, 1513, 1458, 1384, 1303, 1249, 1174, 1094, 822, 742 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 1.17 (3H, d, J=6.4 Hz, C27), 2.57 (11H, ddd, J=16.6, 7.8, 2.4 Hz, C24), 2.67 (11H, ddd, J=16.6, 4.4, 1.6 Hz, C24), 3.71 (1H, dq, J=6.4, 4.5 Hz, C26), 3.78 (3H, s, CH₃O), 4.05 (1H, ddd, J=7.8, 4.5, 4.4 Hz, C25), 4.45 (1H, d, J=11.8 Hz, CH₂Ph), 4.50 (2H, s, CH₂Ph), 4.58 (1H, d, J=11.8 Hz, CH₂Ph), 6.84 (2H, d, J=8.7 Hz, Ar), 7.20-7.35 (7H, m, Ar), 9.70 (1H, dd, J=2.4, 1.6 Hz, CHO); ¹³C NMR (75 MHz, CDCl₃) δ 14.2, 44.1, 55.1, 70.9, 72.0, 74.5, 75.1, 113.8, 127.7, 127.7, 128.4, 129.5, 130.2, 138.4, 159.4, 201.4; HRMS Calcd for C₂₀H₂₄O₄ (M⁺): 328.1675. Found: 328.1664; Anal. Calcd for C₂₀H₂₄O₄: C, 73.13; H, 7.37. Found: C, 72.81; H, 7.40; [α]_(D) ²⁰=−9.1° (c 1.06, CH₂Cl₂).

1B. Diketone 101

A mixture of methylisopropyl ketone (53.4 mL, 0.5 mol) and paraformaldehyde (19.5 g, 0.65 mol) in 200 mL CF₃CO₂H was stirred at 60° C. for 18 h. The reaction mixture was concentrated on a rotary evaporator (warm water bath) with a KOH trap and poured into a cold (5° C.) mixture of EtOAc and sat. aqueous NaHCO₃. The layers were separated and the organic layer was washed with brine, dried over MgSO₄, and concentrated in vacuo. Short path distillation gave the trifluoroacetate ester of 3,3-dimethyl-4-hydroxy butanone (bp=53-55° C. at 2 mm Hg, 72.4 g) in 68% yield. This material was dissolved in 400 mL MeOH and treated with 190 mL of 2N NaOH at 0° C. After 1 h at 0° C., the reaction mixture was concentrated in vacuo and the residue was partitioned between EtOAc and sat. aqueous NH₄Cl. The organic layer was washed with brine, dried over MgSO₄, and concentrated to afford 40.1 g (100%) of 3,3-dimethyl-4-hydroxy butanone (A5) as a colorless liquid. Crude A5 was taken up in 200 mL anhydrous DMF and treated with t-butyldimethylsilylchloride (57.3 g, 0.38 mol) and imidazole (25.9 g, 0.38 mol) at 0° C. After stirring at rt for 2 h, the solution was diluted with EtOAc, washed with sat. aqueous NaHCO₃ solution and brine, dried over MgSO₄ and concentrated in vacuo. The residue was distilled under reduced pressure to afford silyl ether ketone A6 (bp=65° C. at 1.5 mm Hg, 55.76 g, 81%) as a colorless oil.

A6: R_(f) (25% EtOAc/hexanes)=0.90; IR (film) 2957, 2931, 2858, 1713, 1473, 1362, 1257, 1136, 1097, 838, 777 cm⁻¹; ¹H NMR (300 MHZ, CDCl₃) δ 0.03 (6H, s), 0.87 (9H, s), 1.09 (6H, s), 2.17 (3H, s), 3.57 (2H, s); ³C NMR (75 M Hz, CDCl₃) δ 5.7, 18.1, 21.4, 25.7, 26.1, 49.6, 70.2, 213.4; HRMS Calcd for C₁₂H₂₆O₂Si (M⁺−CH₃): 215.1467. Found: 215.1469.

To a stirred solution of diisopropylamine (12.6 mL, 96 mmol) in THF (200 mL) was added n-butyllithium (38.4 mL, 2.5M in hexane, 96 mmol) dropwise at 0° C. The mixture was stirred at 0° C. for 30 min, cooled to −78° C., and treated with a solution of ketone A6 (20.0 g, 87 mmol) in THF (50 mL) slowly over 10 min. After stirring at −78° C. for 40 min, acetaldehyde (5.34 mL, 96 mmol) was added and the mixture was kept at −78° C. for 2 h and at −40° C. for 1.5 h. The reaction was quenched with saturated NH₄Cl solution (20 mL) and allowed to warm gradually to rt. The mixture was extracted with ether and the combined organics were washed with brine, dried over MgSO₄ and concentrated in vacuo to afford nearly pure aldol A7 (19 g, 80%) as a pale yellow oil. An analytical sample was obtained following chromatography on silica gel.

A7: R_(f) (15% EtOAc/hexanes)=0.33; IR (film) 2958, 2930, 2858, 1699, 1472, 1392, 1363, 1258, 1101, 838, 777 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 4.16 (1H, m), 3.55 (2H, s), 3.36 (1H, s), 2.74 (1H, dd, J=18.0, 2.4 Hz), 2.68 (1H, dd, J=18.0, 9.0 Hz), 1.16 (3H, d, J=6.3 Hz), 1.08 (3H, s), 1.07 (3H, s), 0.85 (9H, s), 0.01 (6H, s); ¹³C NMR (75 M Hz, CDCl₃) δ −5.7, 18.1, 21.3, 22.3, 25.8, 46.2, 49.7, 63.9, 70.2, 216.8; Anal. Calcd for C₁₄H₃₀O₃Si: C, 61.26; H, 11.02. Found: C, 61.01; H, 11.28.

Formula 101

To a solution of oxalyl chloride (5.3 mL, 60.4 mmol) in CH₂Cl₂ (150 mL) was added dimethyl sulfoxide (8.56 mL, 120.8 mmol) dropwise at −78° C. After 20 min, a solution of crude alcohol A7 (15.0 g, 54.9 mmol) in CH₂Cl₂ (150 mL) was added over 10 min and the mixture was stirred at −78° C. for 1 h. Et₃N (38 mL, 275 mmol) was added and the mixture was stirred for 20 min, brought to 0° C., quenched with sat. aqueous NH₄Cl and diluted with EtOAc. The layers were separated and the aqueous layer was extracted with EtOAc (2×). The combined organics were washed with brine, dried over MgSO₄, and concentrated in vacuo. Flash chromatography on silica gel (5→10% EtOAc/hexanes) provided enolic β-diketone 101 (12.0 g, 81%) as a yellow oil.

101: R_(f) (15% EtOAc/hexanes)=0.68; IR (film) 2957, 2930, 1606, 1472, 1362, 1257, 1102, 838, 777 cm⁻¹; ¹H NMR (300 MHZ, CDCl₃) δ 0.01 (6H, s), 0.85 (9H, s), 1.10 (6H, s), 2.05 (3H, s), 3.53 (2H, s), 5.62 (1H, s); ¹³C NMR (75 M Hz, CDCl₃) δ −5.5, 18.3, 22.1, 25.5, 25.9, 44.9, 70.0, 98.0, 192.6, 198.5; Anal. Calcd for C₁₄H₂₈O₃Si: C, 61.72; H, 10.36. Found: C, 61.08; H, 10.43.

1C. Dibenzyl Ether Octanoate 111 Formulae 104a and 104b

To a solution of diisopropylamine (3.21 mL, 23 mmol) in 25 mL THF at −60° C. was added n-butyllithium (1.6 M in hexanes, 13.83 mL, 22.13 mmol) dropwise. The colorless solution was warmed to 0° C. and stirred for 30 min. A THF solution (35 mL) of diketone 101 (Example 1B) (2.98 g, 10.9 mmol) was subsequently added via cannula and the mixture was stirred for 1 h at 0° C. The reaction was re-cooled to −78° C. and treated with a solution of aldehyde 102 (Example 1A) (3.0 g, 9.15 mmol) in 35 mL THF. After 30 min at −78° C., the mixture was quenched with sat. NH₄Cl and brought to rt. The aqueous layer was extracted with Et₂O, the combined organics were dried over MgSO₄ and the solvent was removed in vacuo. The crude residue was quickly passed through a column of silica gel (20% EtOAc/hexanes) to provide 5.40 g (98%) of aldol diastereomer mixture 103 as an approximately 1:1 mixture of diastereomers.

A portion of isolated 103 (2.50 g, 4.2 mmol) was dissolved in 60 mL anhydrous toluene and treated with 4 Å molecular sieves (1.55 g) and p-toluenesulfonic acid (60 mg). The reaction was stirred at room temperature for 4.5 h, quenched with 2 mL pyridine and concentrated. The residue was taken up in Et₂O, washed with sat. NaHCO₃, dried over MgSO₄ and the solvent was removed in vacuo. Flash chromatography (20→25% EtOAc/hexanes) afforded pyrone compounds 104a (1.0 g) and 104b (1.2 g) in 90% overall yield as colorless oils.

104b: R_(f) (30% EtOAc/hexanes)=0.59; ¹H NMR (300 M Hz, CDCl₃) δ 0.05 (6H, s, TBS), 0.85 (9H, s, TBS), 1.05 (3H, s, C18 Me), 1.06 (3H, s, C18 Me), 1.20 (3H, d, J=5.8 Hz, C27), 1.98 (2H, m, C24), 2.12 (1H, dd, J=16.7, 3.6 Hz, C24), 2.28 (1H, dd, J=16.7, 13.7 Hz, C22), 3.44 (2H, s, C17), 3.54 (1H, m, C25), 3.72 (1H, m, C26), 3.81 (3H, s, CH₃O), 4.23 (1H, m, C23), 4.40 (1H, d, J=11.2 Hz, CH₂Ph), 4.47 (1H, d, J=11.8 Hz, CH₂Ph), 4.52 (1H, d, J=11.2 Hz, CH₂Ph), 4.63 (1H, d, J=11.8 Hz, CH₂Ph), 5.39 (1H, s, C20), 6.85 (2H, d, J=8.7 Hz, Ar), 7.17-7.35 (7H, m, Ar); ¹³C NMR (75 M Hz, CDCl₃) δ −5.5, 14.7, 18.2, 22.4, 25.9, 34.5, 40.9, 42.4, 55.4, 69.6, 71.2, 72.0, 74.3, 76.2, 103.5, 114.1, 127.9, 128.0, 128.7, 128.8, 129.9, 130.4, 138.8, 159.7, 182.2, 193.7; HRMS Calcd for C₃₄H₅₀O₆Si (M⁺): 582.3377. Found: 582.3370

104a: R_(f) (30% EtOAc/hexanes)=0.52; IR 2955, 2857, 1667, 1599, 1514, 1397, 1336, 1301, 1249, 1174, 1102, 838 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.05 (6H, s, TBS), 0.87 (9H, s, TBS), 1.10 (3H, s, C18 Me), 1.14 (3H, s, C18 Me), 1.21 (3H, d, J=6.3 Hz, C27), 1.71 (1H, m, C24), 2.10 (1H, m, C24), 2.42 (2H, m, C22), 3.48 (1H, d, J=9.3 Hz, C17), 3.58 (1H, d, J=9.3 Hz, C17), 3.76 (1H, m, C26), 3.82 (3H, s, CH₃O), 3.83 (1H, m, C25), 4.43 (1H, d, J=10.7 Hz, CH₂Ph), 4.56 (1H, br m, C23), 4.57 (11H, d, J=11.8 Hz, CH₂Ph), 4.63 (11H, d, J=10.7 Hz, CH₂Ph), 4.65 (11H, d, J=11.8 Hz, CH₂Ph), 5.47 (11H, s, C20), 6.87 (2H, d, J=8.6 Hz, Ar), 7.20 (2H, d, J=8.6 Hz, Ar), 7.32-7.38 (5H, m, Ar); ¹³C-NMR (75 M Hz, CDCl₃) δ −5.6, 14.4, 18.1, 22.3, 22.5, 25.7, 35.2, 41.5, 42.3, 55.2, 69.4, 71.1, 72.8, 74.8, 75.9, 76.4, 103.2, 113.8, 127.5, 128.3, 129.3, 130.3, 138.5, 159.2, 181.1, 193.4; HRMS Calcd for C₃₄H₅₀O₆Si (M⁺): 582.3377. Found: 582.3369; Anal. Calcd for C₃₄H₅₀O₆Si: C, 70.06; H, 8.65. Found: C, 69.95; H, 8.77; [α]_(D) ²⁰=+43.9° (c 0.70, CH₂Cl₂).

Formula 105

To a solution of pyrone 104a (680 mg, 2.4 mmol) and CeCl₃, 7H₂O (218 mg, 0.59 mmol) in 40 mL methanol was added solid NaBH₄ (89 mg, 2.3 mmol) in a single portion at −20° C. The reaction mixture was stirred for 1 h at −20° C. and then quenched with 50 mL brine. The mixture was brought to rt, filtered through a pad of Celite™ and extracted with EtOAc (4×). The combined organics were dried over Na₂SO₄ and concentrated in vacuo to afford a crude allylic alcohol. This moderately stable oil was carried forward without purification.

Crude allylic alcohol was dissolved in 45 mL CH₂Cl₂/MeOH (2:1) and treated with solid NaHCO₃ (243 mg, 2.9 mmol). Purified m-CPBA (377 mg, 2.20 mmol) was added in a single portion and the reaction mixture was stirred at rt for 1 h. The mixture was quenched with Et₃N (15 mL), stirred for 20 min, diluted with 200 mL Et₂O, and filtered through a pad of Celite™. The filtrate was concentrated in vacuo and the residue was purified by flash chromatography (40% EtOAc/hexanes) to give 650 mg (71% from 104a) of syn diol 105 as a colorless oil.

105: R_(f) (30% EtOAc/hexanes)=0.29; IR (film) 3372, 1613, 1514, 1465, 1390, 1302, 1249, 1180, 1150, 1072, 935, 837, 779, 736, 698, 673 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.09 (6H, s, TBS), 0.91 (9H, s, TBS), 1.03 (3H, s, C18 Me), 1.07 (3H, s, C18 Me), 1.18 (3H, d, J=6.3 Hz, C27), 1.50-1.68 (2H, m, C24/C22), 1.80 (1H, m, C22), 2.57 (1H, app d, J=11.2 Hz, C24), 3.28 (3H, s, CH₃O), 3.39, (1H, d, J=10.1 Hz, C17), 3.62 (1H, d, J=11.2 Hz, C17), 3.79 (3H, s, CH₃O), 3.71-3.94 (5H, m, CH₂Ph, C23/C25/C26), 4.40 (1H, d, J=10.7 Hz, CH₂Ph), 4.56 (1H, d, J=11.8 Hz, CH₂Ph), 4.62 (1H, d, J=11.5 Hz, CH₂Ph), 5.40 (1H, d, J=2.5 Hz, OH), 6.84 (2H, d, J=8.7 Hz, PMB), 7.18 (2H, d, J=8.7 Hz, PMB), 7.37-7.26 (5H, m, Bn).

Formula 106

A solution of diol 105 (0.81 g, 1.28 mmol) and 4-dimethylaminopyridine (DMAP, 0.55 g, 4.48 mmol) in 22 mL CH₂Cl₂ was cooled to −10° C. and treated with benzoyl chloride (193 μL, 1.66 mmol) dropwise via syringe. The resulting mixture was stirred at −10° C. for 30 min, quenched with sat NaHCO₃ and diluted with EtOAc (150 mL). The organic layer was washed with H₂O and brine, dried over Na₂SO₄ and concentrated in vacuo to afford a crude mixture of C21 monobenzoate and 4-dimethylaminopyridine as a colorless paste.

The crude C21 monobenzoate was taken up in 45 mL CH₂Cl₂ and treated with solid 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (Dess-Martin periodinane or DMP, 1.78 g, 4.20 mmol) at rt. The solution was stirred for 10 h at rt after which a second portion (0.50 g, 1.18 mmol) of DMP was added. The opaque white mixture was stirred for another 1.5 h and quenched with 30 mL sat. NaHCO₃/Na₂S₂O₃. The two phase system was vigorously stirred until the organic layer cleared (˜25 min). The layers were separated and the aqueous phase was extracted with CH₂Cl₂ (2×). The combined organics were dried over Na₂SO₄ and concentrated in vacuo to provide a colorless semi-solid. Flash chromatography on silica gel (25% EtOAc/hexanes) gave 106 (0.85 g-90% from 105) as a colorless oil.

106: R_(f) (15% EtOAc/hexanes)=0.43; IR (film) 2954, 2933, 1754, 1723, 1610, 1513, 1451, 1267, 1251 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.01 (6H, s, TBS), 0.88 (9H, s, TBS), 1.12 (3H, s, C18 Me), 1.14 (3H, s, C18 Me), 1.19 (3H, d, J=6.3 Hz, C27), 1.64-1.73 (11H, m, C24), 1.82-1.90 (1H, m, C24), 2.13 (1H, app q, J=12.4 Hz, C22), 2.40 (1H, ddd, J=12.4, 6.5, 1.6 Hz, C22), 3.54 (3H, s, C19 OCH₃), 3.65 (1H, d, J=9.2 Hz, C17), 3.69 (1H, d, J=9.2 Hz, C17) 3.72-3.81 (2H, m), 3.81 (3H, s, ArOCH₃), 3.89 (1H, m), 4.49 (1H, d, J=10.7 Hz, CH₂Ar), 4.57 (1H, d, J=13.0 Hz, CH₂Ar), 4.61 (1H, d, J=13.0 Hz, CH₂Ar), 4.62 (1H, d, J=10.7 Hz, CH₂Ar), 5.80 (1H, dd, J=12.9, 6.3 Hz, C21), 6.87 (2H, d, J=8.3 Hz, Ar), 7.26-7.36 (7H, m, Ar), 7.45 (2H, m, Ar), 7.58 (1H, app t, J=7.2 Hz, Ar), 8.09 (2H, d, J=7.2 Hz, Ar); ¹³C-NMR (75 M Hz, CDCl₃) δ −5.3, 14.7, 18.5, 20.4, 20.6, 26.1, 35.5, 40.4, 45.1, 53.8, 55.5, 65.4, 67.2, 71.4, 73.0, 73.3, 75.1, 103.8, 114.1, 127.9, 128.6, 128.7, 129.7, 129.9, 130.2, 131.0, 133.6, 138.9, 159.6, 165.9, 198.6; HRMS Calcd for C₄₂H₅₈O₉Si (M⁺−MeOH): 702.3588. Found: 702.3563; Anal. Calcd for C₄₂H₅₈O₉Si: C, 68.63; H, 7.96. Found: C, 68.28; H, 8.11; [α]_(D) ²⁰=+22.4° (c 1.53, CH₂Cl₂).

Formula 107

A solution of benzoate 106 (0.85 g, 1.16 mmol) in 20 mL THF/MeOH (3:1) was titrated with SmI₂ (0.1 M in THF, 25.5 mL, 2.55 mmol) at −78° C. until an olive green color persisted. The reaction mixture was quenched with 4 mL sat. NaHCO₃, warmed to rt and diluted with EtOAc (150 mL). The organic layer was washed with NaHCO₃, H₂O and brine, dried over Na₂SO₄ and concentrated in vacuo. Flash chromatography on silica gel (20% EtOAc/hexanes) afforded ketone 107 (675 mg, 95%) as a light yellow oil.

107: R_(f) (15% EtOAc/hexanes)=0.39; IR (film) 2954, 2930, 1723, 1612, 1514, 1464, 1250, 1088 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.01 (6H, s, TBS), 0.87 (9H, s, TBS), 0.95 (3H, s, C18 Me), 1.05 (3H, s, C18 Me), 1.19 (3H, d, J=6.3 Hz, C27), 1.64 (1H, m, C24), 1.85-1.96 (3H, m, C21+C24), 2.29 (1H, 5 line m, C22), 2.65 (1H, m, C22), 3.23 (3H, s, C19 OCH₃), 3.31 (1H, d, J=9.9 Hz, C17), 3.71 (1H, d, J=9.9 Hz, C17), 3.78 (3H, s, ArOCH₃), 3.79 (11H, m), 3.98 (11H, m), 4.17 (11H, m), 4.43 (11H, d, J=11.0 Hz, CH₂Ar), 4.58 (2H, s, CH₂Ar), 4.60 (11H, d, J=11.0 Hz, CH₂Ar), 6.84 (2H, d, J=8.7 Hz, PMB), 7.19 (2H, d, J=8.7 Hz, PMB), 7.28-7.35 (5H, m, Bn); ¹³C NMR (75 M Hz, CDCl₃) δ −5.5, −5.4, 14.5, 18.6, 19.8, 20.2, 26.0, 29.7, 36.5, 38.2, 46.2, 52.4, 55.4, 68.6, 70.6, 71.3, 72.3, 74.7, 103.6, 114.1, 127.9, 128.7, 129.6, 131.0, 138.9, 159.5, 207.5; HRMS Calcd for C₃₅H₅₄O₇Si (M⁺−MeOH): 582.3377. Found: 582.3372; Anal. Calcd for C₃₅H₅₄O₇Si: C, 68.37; H, 8.85. Found: C, 68.04; H, 8.84; [α]_(D) ²⁰=+21.9° (c 0.72, CH₂Cl₂).

To a solution of diisopropylamine (150 ΦL, 1.15 mmol) in THF (1.61 mL) was added n-BuLi (2.5 M in hexanes, 440 ΦL, 1.10 mmol) dropwise at 0° C. After 5 min at 0° C., a 1.81 mL aliquot (0.5 M LDA, 0.90 mmol) was removed via syringe and slowly added to a solution of ketone 107 (483 mg, 0.79 mmol) in THF (20 mL) at −78° C. The solution was stirred for 10 min, treated with a stock solution of OHCCO₂Me (0.5 M in Et₂O, 4.0 mL, 2.0 mmol), kept at −78° C. for 20 min and quenched with 3 mL sat. NH₄Cl. The reaction mixture was brought to rt and diluted with 200 mL EtOAc. The organic layer was washed with H₂O (2×) and brine, dried over Na₂SO₄ and concentrated in vacuo. The crude residue was chromatographed on silica gel (35% EtOAc/hexanes) to afford residual 107 (142 mg) and aldols 108 as a mixture of diastereomers (352 mg, 90% based on recovered 107).

The isolated 108 mixture and Et₃N (418 ΦL, 3.0 mmol) were dissolved in anhydrous CH₂Cl₂ (15 mL) and cooled to −10° C. Methanesulfonylchloride (116 ΦL, 1.5 mmol) was added via syringe and the solution was stirred at −10° C. for 30 min. 5 mL sat. NaHCO₃ was added, the reaction mixture was warmed to rt and diluted with 100 mL EtOAc. The organic layer was washed with H₂O and brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was immediately dissolved in THF (30 mL) and treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 75 ΦL, 0.50 mmol) dropwise at rt. The resulting bright yellow solution was stirred at rt for 20 min, treated with sat. NH₄Cl and diluted with 150 mL EtOAc. The organic layer was washed with H₂O and brine, dried over Na₂SO₄ and concentrated in vacuo to afford an orange residue which was chromatographed on silica gel (20% EtOAc/hexanes) to afford exocyclic methacrylate (enone) 109.1 (267 mg, 78% from 108) as a yellow oil.

109.1: R_(f) (30% EtOAc/hexanes)=0.63; IR (film) 2954, 2930, 1724, 1707, 1612, 1514, 1464, 1250, 1088 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.05 (6H, s, TBS), 0.81 (9H, s, TBS), 0.91 (3H, s, C18 Me), 1.00 (3H, s, C18 Me), 1.19 (3H, d, J=6.4 Hz, C27), 1.74 (1H, m, C24), 1.94 (1H, m, C24), 2.71 (1H, ddd, J=17.6, 12.4, 3.1 Hz, C22), 3.19 (3H, s, C19 OCH₃), 3.26 (1H, d, J=10.0 Hz, C17), 3.49 (1H, d, J=17.6 Hz, C22), 3.68 (1H, d, J=10.0 Hz, C17), 3.74 (3H, s), 3.77 (3H, s), 3.80 (1H, m), 3.97 (1H, m), 4.19 (1H, m), 4.40 (1H, d, J=10.9 Hz, CH₂Ar), 4.53-4.63 (3H, m, CH₂Ar), 6.64 (1H, d, J=1.9 Hz, C34), 6.82 (2H, d, J=8.7 Hz, PMB), 7.14 (2H, d, J=8.7 Hz, PMB), 7.27-7.36 (5H, m, Bn); ¹³C-NMR (75 M Hz, CDCl₃) δ −5.9, −5.8, 14.1, 18.3, 19.3, 19.8, 25.7, 35.2, 36.0, 46.6, 51.6, 52.1, 55.1, 68.2, 69.5, 71.0, 71.7, 74.2, 76.0, 104.0, 113.8, 122.4, 127.6, 128.4, 129.1, 130.6, 138.6, 147.3, 159.2, 166.4, 196.2; HRMS Calcd for C₃₈H₅₆O₉Si (M⁺−MeOH): 652.3433. Found: 652.3435; Anal. Calcd for C₃₈H₅₆O₉Si: C, 66.64; H, 8.24. Found: C, 66.87; H, 8.13; [α]_(D) ²⁰=−55.8° (c 0.78, CH₂Cl₂).

To a solution of enone 109.1 (502 mg, 0.734 mmol) and CeCl₃.7H₂O (137 mg, 0.367 mmol) in methanol (23 mL) was added solid NaBH₄ (56 mg, 1.47 mmol) in a single portion at −30° C. Rapid gas evolution subsided after 3 min. After an additional 30 min at −30° C., the reaction mixture was poured directly onto a silica gel column and the product quickly eluted with 25% EtOAc/hexanes to afford the corresponding axial alcohol 110.1 (478 mg) as a colorless oil.

Octanoic acid (232 mg, 1.61 mmol) and Et₃N (292 μL, 2.20 mmol) were dissolved in 20 mL toluene and treated with 2,4,6-trichlorobenzoylchloride (230 μL, 1.47 mmol) dropwise at rt. After 1 h at rt, a toluene solution (7 mL) of freshly prepared 110.1 was added gradually via syringe and stirring was continued for 40 min. The reaction mixture was quenched with 10 mL sat. NaHCO₃, diluted with EtOAc and washed successively with sat. NH₄Cl and brine. The organics were dried over Na₂SO₄, the solvent was removed in vacuo, and the residue was chromatographed on silica gel (25% EtOAc/hexanes) to provide octanoate 111.1 as a colorless oil (551 mg, 93% from 109.1).

111.1: R_(f) (25% Et₂O/hexanes)=0.33; IR (film) 2928, 2857, 1747, 1722, 1667, 1614, 1514, 1463, 1250, 1155, 1081, 836.2 cm⁻¹; ¹H NMR (300M Hz, CDCl₃) 6-0.01 (6H, s, TBS), 0.88 (12H, br s, TBS+octanoate Me), 0.99 (3H, s, C18 Me), 1.03 (3H, s, C18 Me), 1.19 (3H, d, J=6.3 Hz, C27), 1.20-1.35 (8H, m), 1.60-1.80 (3H, m), 1.89 (1H, m, C24), 2.31-2.40 (3H, m), 3.26 (3H, s, C19 OCH₃), 3.44 (1H, dd, J=15.6, 1.8 Hz, C22), 3.56 (1H, d, J=9.3 Hz, C17), 3.60 (1H, d, J=9.3 Hz, C17), 3.68 (3H, s), 3.78 (1H, m), 3.79 (3H, s), 3.93 (1H, dd, J=8.4, 4.8 Hz), 4.13 (1H, m), 4.38 (1H, d, J=10.8 Hz, CH₂Ar), 4.57 (1H, d, J=10.8 Hz, CH₂Ar), 4.60 (2H, s, CH₂Ar), 5.57 (1H, s, C20), 5.89 (1H, s, C34), 6.83 (2H, d, J=8.4 Hz, PMB), 7.16 (2H, d, J=8.4 Hz, PMB), 7.28-7.38 (5H, m, Bn); ¹³C NMR (75 M Hz, CDCl₃) δ −5.4, 14.1, 14.2, 14.4, 18.4, 20.7, 22.7, 24.8, 26.0, 28.9, 26.0, 28.9, 31.7, 33.2, 34.6, 36.4, 47.1, 51.2, 55.3, 67.6, 68.1, 71.2, 72.2, 74.7, 76.5, 103.1, 114.0, 117.0, 127.8, 128.6, 129.5, 129.9, 130.9, 139.0, 153.1, 159.5, 166.7, 172.2; HRMS Calcd for C₄₆H₇₂O₁₀Si (M⁺−MeOH): 780.4632. Found: 780.4610; [α]_(D) ²⁰=−5.1° (c 1.80, CH₂Cl₂).

Example 2 Exemplary Linkers 2A. Ketal Acid 406 Formula 402

To a stirred solution of the 1,3 menthone acetal of 1,3,5-pentanetriol 401 (3.33 g, 13 mmol) in 23 mL of anhydrous CH₂Cl₂ was added 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (Dess-Martin periodinane, 6.60 g, 15.6 mmol) in a single portion. The mixture was stirred at rt for 30 min, poured onto a column of silica gel and the product eluted with 15% EtOAc/hexanes to afford 3.013 g (90%) of pure aldehyde 402 as a colorless oil.

402: R_(f) (20% EtOAc/hexanes)=0.50; IR (film) 2952, 2869, 1728, 1456, 1383, 1308, 1265 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 9.80 (1H, dd, J=1.8, 2.5 Hz), 4.36 (1H, dddd, J=2.9, 4.3, 7.4, 8.2 Hz), 4.13 (1H, ddd, J=2.7, 11.7, 11.9 Hz), 3.83 (1H, ddd, J=1.3, 5.2, 11.7 Hz), 2.72 (1H, ddd, J=1.9, 3.1, 13.5 Hz), 2.56 (1H, ddd, J=2.5, 8.2, 16.1 Hz), 2.44 (1H, ddd, J=1.8, 4.3, 16.1 Hz), 2.39 (1H, dsept, J=1.6, 7.1 Hz), 1.54-1.76 (3H, m), 1.29-1.53 (4H, m), 1.17 (1H, ddd, J=1.9, 4.1, 12.4 Hz), 0.90 (3H, d, J=6.3 Hz), 0.87 (3H, d, J=7.0 Hz), 0.85 (3H, d, J=7.0 Hz), 0.72 (1H, t, J=13.2 Hz); ¹³C NMR (75 M Hz, CDCl₃) δ 201.4, 100.9, 63.8, 58.7, 51.1, 49.9, 37.2, 34.8, 31.1, 28.9, 24.2, 23.7, 22.2, 21.7, 18.8; HRMS Calc'd for C₁₅H₂₆O₃: 254.1882. Found: 254.1877; [α]_(D) ²⁰=−11.2° (c 1.28, CHCl₃).

Formulae 403a and 403b

To a stirred solution of aldehyde 402 (3.013 g, 11.86 mmol) in 40 mL of anhydrous CH₂Cl₂ was added a solution of (+)-Eu(hfc)₃ (1.42 g, 1.19 mmol) in CH₂Cl₂ (16 mL) at rt. The resultant clear yellow solution was stirred for 5 min before 1-methoxy-3-(trimethyl-silyloxy)-1,3-butadiene (3.47 mL, 3.07 g, 17.8 mmol) was introduced via syringe. The yellow solution was stirred at rt for 20 h, treated with 0.5 mL CF₃CO₂H and stirred for an additional 15 min. The solution was quenched with sat. NaHCO₃, diluted with EtOAc (200 mL), washed with H₂O and brine, dried over MgSO₄ and concentrated in vacuo. Purification by flash chromatography (20->25% EtOAc/hexanes) provided anti pyrone 403b (2.53 g, 66%) and syn pyrone 403a (1.30 g, 34%) as colorless solids.

403b: mp=113-114° C. (hexanes); R_(f) (15% EtOAc/hexanes)=0.25; ¹H NMR (300 M Hz, CDCl₃) δ 7.31 (1H, d, J=6.0 Hz), 5.40 (1H, d, J=6.0 Hz), 4.67 (1H, dddd, J=3.0, 5.8, 9.6, 11.8 Hz), 4.03-4.15 (2H, m), 3.81 (1H, ddd, J=1.2, 5.2, 11.6 Hz), 2.70 (1H, ddd, J=1.9, 2.8, 13.5 Hz), 2.42-2.56 (2H, m), 2.38 (1H, dsept, J=1.6, 6.9 Hz), 1.91 (1H, ddd, J=2.3, 9.6, 14.3 Hz), 1.28-1.75 (8H, m), 1.18 (1H, ddd, J=1.9, 3.9, 12.5 Hz), 0.88 (3H, d, J=6.6 Hz), 0.87 (3H, d, J=7.1 Hz), 0.83 (3H, d, J=6.9 Hz), 0.70 (1H, t, J=12.6 Hz); ¹³C NMR (75 M Hz, CDCl₃) δ 192.7, 162.9, 107.3, 100.7, 75.7, 63.3, 58.9, 51.1, 42.5, 41.7, 37.3, 34.8, 31.7, 29.0, 24.2, 23.6, 22.2, 21.9, 18.9; LRMS (EI): 322 (68), 307 (22), 265 (33), 237 (67), 153 (24), 191 (94), 139 (52), 112 (20), 97 (100), 83 (31), 81 (47), 71 (24), 69 (35); HRMS Calcd for C₁₉H₃₀O₄: 322.2144. Found: 322.2142; Anal. Calcd for C₁₉H₃₀O₄: C, 70.77; H, 9.38. Found: C, 70.91; H, 9.58; [α]_(D) ²⁰=+42.5° (c 1.59, CH₂Cl₂).

403a: R_(f) (15% EtOAc/hexanes)=0.15; IR 2952, 2869, 1682, 1597, 1456, 1405, 1269 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 7.33 (1H, d, J=6.0 Hz), 5.40 (1H, dd, J=0.9, 6.0 Hz), 4.61 (1H, dddd, J=2.8, 11.6, 12.0 Hz), 4.09 (1H, ddd, J=2.8, 11.6, 12.0 Hz), 4.00 (1H, dddd, J=1.0, 4.2, 8.3, 15.5 Hz), 3.81 (1H, ddd, J=1.4, 5.4, 11.6 Hz), 2.67 (1H, ddd, J=1.9, 3.2, 13.5 Hz), 2.63 (1H, dd, J=13.3, 16.8 Hz), 2.47 (1H, ddd, J=0.9, 4.0, 16.8 Hz), 2.39 (1H, dsept, J=1.6, 6.9 Hz), 2.02 (1H, ddd, J=5.6, 8.3, 14.0 Hz), 1.79 (1H, ddd, J=4.2, 6.9, 14.0 Hz), 1.20-1.75 (7H, m), 1.17 (1H, ddd, J=1.9, 3.8, 12.6 Hz), 0.89 (3H, d, J=6.7 Hz), 0.87 (3H, d, J=6.9 Hz), 0.86 (3H, d, J=6.9 Hz), 0.70 (1H, t, J=13.5 Hz); ¹³C NMR (75 M Hz, CDCl₃) δ 192.6, 163.3, 107.1, 100.7, 76.4, 63.9, 58.8, 51.1, 41.6, 40.9, 37.3, 34.7, 31.4, 29.3, 24.2, 23.7, 22.2, 21.8, 18.8; LRMS (EI): 322 (61), 307 (18), 265 (27), 237 (56), 151 (21), 139 (33), 112 (17), 97 (100), 83 (26), 81 (34), 71 (17), 69 (60); HRMS Calcd for C₁₉H₃₀O₄: 322.2144. Found: 322.2146; [α]_(D) ²⁰=−57.8° (c 1.5, CH₂Cl₂).

Formula 404

To a stirred solution of pyrone 403a (1.30 g, 4.04 mmol) in 40 mL of anhydrous MeOH was added CeCl₃.7H₂O (904 mg, 2.43 mmol) at rt. After stirring for 10 min, the mixture was cooled to −40° C. and NaBH₄ (306 mg, 8.09 mmol) was added in one portion. The mixture was stirred for an additional 15 min before quenching with a 1:1 mixture of brine and H₂O. The aqueous layer was extracted with EtOAc (4×). The combined organics were washed with sat. NaHCO₃ and brine, dried over Na₂SO₄ and concentrated to afford a clear oil. Purification by flash chromatography (30% EtOAc/hexanes containing 1% Et₃N) afforded unstable allylic alcohol 404 (1.08 g, 82%) as a single diastereomer.

404: IR (film) 3386, 2951, 2869, 1643, 1456, 1380, 1307, 1268, 1231 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 6.35 (1H, br d, J=5.4 Hz), 4.75 (1H, ddd, J=1.9, 1.9, 6.1 Hz), 4.36-4.46 (1H, m), 4.08-4.17 (1H, m), 4.08 (1H, ddd, J=2.7, 12.4, 12.4 Hz), 3.98 (1H, dddd, J=2.7, 5.2, 7.9, 10.9 Hz), 3.81 (1H, ddd, J=1.5, 5.2, 11.5 Hz), 2.69 (1H, br d, J=13.5 Hz), 2.39 (1H, dsept, J=1.7, 6.9 Hz), 2.18 (1H, dddd, J=1.7, 1.9, 6.4, 13.0 Hz), 1.90 (1H, ddd, J=6.6, 7.7, 14.0 Hz), 1.33-1.74 (9H, m), 1.17 (1H, ddd, J=1.9, 4.4, 12.3 Hz), 0.90 (3H, d, J=6.2 Hz), 0.88 (6H, d, J=6.9 Hz), 0.69 (1H, t, J=12.7 Hz); ¹³C NMR (75 M Hz, CDCl₃) δ 145.3, 105.5, 100.6, 71.4, 64.3, 63.0, 58.9, 51.2, 41.5, 37.7, 37.4, 34.9, 31.3, 29.2, 24.2, 23.7, 22.2, 21.8, 18.8; [α]_(D) ²⁰=+2.0° (c 0.59, CHCl₃).

Formula 405

To a solution of 1.08 g (3.32 mmol) of allylic alcohol 404 in 66 mL of ethylvinylether was added mercury(II)trifluoroacetate (142 mg, 0.33 mmol) in a single portion at −10° C. The resulting colorless solution was stirred for 20 h at 5° C., diluted with Et₂O, washed with sat. NaHCO₃ and brine, dried over Na₂SO₄, and concentrated in vacuo to give a clear, colorless oil. Rapid flash chromatography (10% EtOAc/hexanes containing 1% Et₃N) afforded 865 mg (74%) of the corresponding allyl vinyl ether along with 208 mg (19%) of recovered 404. The vinyl ether was immediately dissolved in 100 mL n-nonane and heated at 145° C. for 3.5 h. The solution was cooled to 70-80° C. and the solvent was removed by short path distillation at reduced pressure. The remaining residue was purified by flash chromatography (10% EtOAc/hexanes) to provide Claisen product 405 (612 mg, 71%) as a colorless oil.

405: R_(f) (30% EtOAc/hexanes)=0.75; IR (film) 2950, 2869, 1728, 1646, 1456, 1373, 1307, 1267 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 9.78 (1H, t, J=2.4 Hz), 5.89 (1H, dddd, J=2.0, 2.0, 4.7, 10.0 Hz), 5.64 (1H, dddd, J=1.3, 1.3, 2.5, 10.0 Hz), 4.57-4.66 (1H, m), 4.08 (1H, ddd, J=2.7, 12.1, 12.1 Hz), 3.90-4.01 (1H, m), 3.66-3.82 (2H, m), 2.69 (1H, ddd, J=1.9, 3.0, 13.4 Hz), 2.55 (2H, dd, J=2.4, 6.2 Hz), 2.40 (1H, dsept, J=1.6, 6.9 Hz), 1.12-2.14 (12H, m), 0.89 (3H, d, J=6.6 Hz), 0.88 (6H, d, J=7.1 Hz), 0.68 (1H, t, J=12.9 Hz); ¹³C NMR (75 M Hz, CDCl₃) δ 201.7, 128.5, 126.3, 100.5, 70.6, 70.5, 64.4, 59.1, 51.2, 48.5, 42.5, 37.4, 34.9, 31.5, 30.7, 29.1, 24.3, 23.7, 22.2, 21.9, 18.9; MS (EI) 350 (51), 335 (45), 322 (14), 293 (65), 279 (15), 265 (52), 255 (15), 139 (17), 135 (29), 97 (28), 83 (43), 81 (100), 79 (17), 69 (27), 67 (24); HRMS Calcd for C₂₁H₃₄O₄: 350.2457. Found: 350.2466; [α]_(D) ²⁷=0° (CH₂Cl₂).

Formula 406

To a stirred mixture of trimethylsilyl diethylphosphonoacetate (72 μl, 76.7 mg, 0.286 mmol) in anhydrous THF (1 mL) was added of n-butyllithium (2.5 M in hexanes, 109 μL, 0.272 mmol) dropwise at −78° C. After stirring for 20 min at −78° C. and 15 min at rt, the mixture was cooled to −78° C. and treated with a THF solution (1 mL) of aldehyde 405 (45 mg, 0.129 mmol). The mixture was allowed to warm to rt over 2 h and stirring was continued for 1 h. The mixture was diluted with 50 mL EtOAc and acidified with 10 mL of a 0.1 N aqueous NaHSO₄ solution. The phases were separated and the aqueous phase was extracted with EtOAc (3×). The combined organic extracts were washed with brine, dried over MgSO₄, and concentrated to give a colorless oil. Rapid filtration through a short pad of silica gel (20% acetone/benzene) gave a crude product which was dissolved in 2 mL methanol. Palladium on activated charcoal (10%, 5 mg) was added and the mixture was stirred at room temperature for 18 h under balloon pressure of hydrogen gas. Filtration through Celite™ and flash chromatography on silica gel (40% EtOAc/hexanes containing 1% acetic acid) gave 21.8 mg (0.055 mmol, 43%) of ketal carboxylic acid 406 as a clear, colorless oil. R_(f) (40% EtOAc/hexanes+1% AcOH)=0.44; IR (film) 3500-2500, 2938, 2868, 1711, 1456, 1377, 1307, 1268, 1158, 1113 cm¹; ¹H NMR (300 M Hz, CDCl₃) δ 4.08 (ddd, 1H, J=2.6, 12.4, 12.4 Hz), 3.98 (dddd, 1H, J=2.4, 6.6, 6.6, 10.7 Hz), 3.81 (ddd, 1H, J=1.2, 5.2, 11.5 Hz), 3.38-3.47 (m, 1H), 3.21-3.31 (m, 1H), 2.70 (br d, 1H, J=12.5 Hz), 2.29-2.47 (m, 3H), 1.62-1.86 (m, 5H), 1.34-1.62 (m, 11H), 1.07-1.30 (m, 3H), 0.78-0.98 (m, 1H), 0.88 (d, 6H, J=7.2 Hz), 0.88 (d, 3H, J=7.0 Hz), 0.67 (t, 1H, J=12.9 Hz); ¹³C NMR (75 M Hz, CDCl₃) δ 179.1, 100.5, 74.2, 64.6, 59.2, 51.2, 42.9, 37.3, 35.4, 34.9, 33.7, 31.50, 31.46, 31.3, 29.0, 24.2, 23.7, 23.6, 22.2, 21.8, 20.9, 18.8; HRMS Calcd for C₂₃H₄₀O₅: 396.2876. Found: 396.2870; [α]²⁸ _(D)=−10.5° (c 1.66, CHCl₃).

2B. Ketal Acid 408 Formula 407

To a solution of the aldehyde 405 (Example 2A, 274 mg, 0.778 mmol) in ethyl acetate (5 mL) was added Pd/C (10 mg) and the atmosphere was exchanged to hydrogen, which was applied for 30 min. The mixture was filtered and concentrated to give the corresponding crude saturated aldehyde, which was directly used in the next step.

A stock solution of Ipc₂B(allyl) was prepared by first dissolving (−)-Ipc₂BOMe (700 mg, 2.22 mmol) in ether (4.15 mL) at 0° C. and adding 1M allyl magnesium bromide (1.78 mL, 1.78 mmol). The mixture was warmed to rt and stirred for 30 min. In a separate flask, the aldehyde was dissolved in ether (4 mL) and treated with the stock solution of Ipc₂B(allyl) (0.3 M, 3.9 mL, 1.17 mmol) at −78° C. After stirring for 2 h at −78° C., the mixture was treated with hydrogen peroxide (30%, 2 mL) and sodium hydroxide (15%, 2 mL) and warmed to rt. After another 2 h, the mixture was partitioned between ethyl acetate and brine. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, and concentrated to give the corresponding crude homoallylic alcohol, which was directly used in the next step.

To a solution of the crude homoallylic alcohol in methylene chloride (5 mL) at 0° C. was added TBSOTf (534 μL, 2.33 mmol) and diisopropyl ethyl amine (676 μL, 3.89 mmol) and stirred for 30 min. The mixture was directly loaded onto silica gel and purified to give the corresponding alkene (237 mg, 60% in 3 steps).

To a solution of the alkene (10 mg, 0.0197 mmol) in tert-butanol (1 mL) at rt was added a solution of KMnO₄ (0.6 mg, 0.0039 mmol) and NaIO₄ (17 mg, 0.0788 mmol) in water (buffered pH 7, 1 mL). After 30 min, the reaction mixture was quenched with sodium thiosulfate. The mixture was partitioned between ethyl acetate and brine. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, and concentrated. Column chromatography afforded TBS ether 407 (6.3 mg, 71%).

407: R_(f)=0.20 (25% ethyl acetate/hexane); [α]²⁵ _(D)=22.6° (c 0.58, CH₂Cl₂); IR (neat)=2933, 1712, 1457, 1255, 1114 cm¹; ¹H NMR (400 M Hz, CDCl₃) δ 4.32 (m, 1H), 4.08 (m, 1H), 3.93 (m, 1H), 3.81 (m, 1H), 3.44 (m, 2H), 2.69 (d, J=13.2 Hz, 1H), 2.62 (dd, J=15.2, 5.2 Hz, 1H), 2.48 (dd, J=15.2, 5.2 Hz, 1H), 2.40 (quint, J=6.8 Hz, 1H), 1.85-1.15 (m, 20H), 0.88 (br s, 18H), 0.12 (s, 3H), 0.11 (s, 3H); ¹³C NMR (100 M Hz, CDCl₃) δ 173.48, 100.44, 73.88, 73.52, 66.15, 64.26, 59.14, 51.24, 43.89, 43.25, 42.48, 37.43, 43.97, 32.13, 31.89, 30.93, 29.23, 25.74, 24.31, 23.77, 23.52, 22.25, 21.89, 19.15, 17.92, −4.77; HRMS: calcd for (C₂₉H₅₄O₆Si)=526.3689. found (M)=526.3687.

Formula 408

To a solution of the aldehyde 405 (Example 2A, 274 mg, 0.778 mmol) in ethyl acetate (5 mL) was added Pd/C (10 mg) and the atmosphere was exchanged to hydrogen, which was applied for 30 min. The mixture was filtered and concentrated to give the corresponding crude saturated aldehyde, which was directly used in the next step.

A stock solution of Ipc2B(allyl) was prepared by first dissolving (−)-Ipc₂BOMe (700 mg, 2.22 mmol) in ether (4.15 mL) at 0° C. and adding IM allyl magnesium bromide (1.78 mL, 1.78 mmol). The mixture was warmed to rt and stirred for 30 min. In a separate flask, the aldehyde was dissolved in ether (4 mL) and treated with the stock solution of Ipc₂B(allyl) (0.3 M, 3.9 mL, 1.17 mmol) at −78° C. After stirring for 2 h at −78° C., the mixture was treated with hydrogen peroxide (30%, 2 mL) and sodium hydroxide (15%, 2 mL) and warmed to rt. After another 2 h, the mixture was partitioned between ethyl acetate and brine. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, and concentrated to give the corresponding crude homoallylic alcohol, which was directly used in the next step.

To a solution of the crude homoallylic alcohol in methylene chloride (5 mL) at 0° C. was added TBSOTf (534 μL, 2.33 mmol) and diisopropyl ethyl amine (676 μL, 3.89 mmol) and stirred for 30 min. The mixture was directly loaded onto silica gel and purified to give the corresponding alkene (237 mg, 60% in 3 steps).

To a solution of the alkene (10 mg, 0.0197 mmol) in tert-butanol (1 mL) at rt was added a solution of KMnO₄ (0.6 mg, 0.0039 mmol) and NaIO₄ (17 mg, 0.0788 mmol) in water (buffered pH 7, 1 mL). After 30 min, the reaction mixture was quenched with sodium thiosulfate. The mixture was partitioned between ethyl acetate and brine. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, and concentrated. Column chromatography afforded TBS ether 407 (6.3 mg, 71%).

407: R_(f)=0.20 (25% ethyl acetate/hexane); [α]²⁵ _(D)=22.6° (c 0.58, CH₂Cl₂); IR (neat)=2933, 1712, 1457, 1255, 1114 cm¹; ¹H NMR (400 M Hz, CDCl₃) δ 4.32 (m, 1H), 4.08 (m, 1H), 3.93 (m, 1H), 3.81 (m, 1H), 3.44 (m, 2H), 2.69 (d, J=13.2 Hz, 1H), 2.62 (dd, J=15.2, 5.2 Hz, 1H), 2.48 (dd, J=15.2, 5.2 Hz, 1H), 2.40 (quint, J=6.8 Hz, 1H), 1.85-1.15 (m, 20H), 0.88 (br s, 18H), 0.12 (s, 3H), 0.11 (s, 3H); ¹³C NMR (100 M Hz, CDCl₃) δ 173.48, 100.44, 73.88, 73.52, 66.15, 64.26, 59.14, 51.24, 43.89, 43.25, 42.48, 37.43, 43.97, 32.13, 31.89, 30.93, 29.23, 25.74, 24.31, 23.77, 23.52, 22.25, 21.89, 19.15, 17.92, −4.77; HRMS: calcd for (C₂₉H₅₄O₆Si)=526.3689. found (M)=526.3687.

2C. 9-Hydroxy-9-t-Butyl L3 Linker Synthon 504 Formula 501

To a solution of aldehyde 402 (Example 2A supra, 803 mg, 3.16 mmol) in ether (10 mL) was added a solution of 4-pentenyl magnesium bromide (0.8M in ether, 4.74 mL, 3.79 mmol) at −78° C. and the mixture was stirred for 30 min. The reaction was quenched with aq. sat. NH₄Cl (10 mL) and allowed to warm to rt. The mixture was then extracted with EtOAc (3×10 mL) and the combined organics were dried over MgSO₄, and concentrated in vacuo. The resulting residue was dissolved in CH₂Cl₂ (15 mL) and Dess-Martin Periodinane (2.02 g, 4.74 mmol) was added at rt. After 3 hours, sat. aq. Na₂SO₃ (10 mL) and sat. aq. NaHCO₃ (10 mL) was added. The mixture was extracted with CHCl₃ (3×10 mL), washed with sat. aq. NaHCO₃ (10 mL), dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography (90% EtOAc/hexane) to obtain ketone 501 (730 mg, 71.6%) as a colorless oil. 501: IR (film) 2957, 2362, 1716, 1637 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) 6, 0.62-0.92 (11H, m), 1.12-1.70 (9H, m) 2.03 (2H, dd, J=14.77, 7.63 Hz), 2.24-2.71 (6H, m), 3.79 (1H, ddd, J=11.53, 5.22, 1.37 Hz), 4.10 (1H, td, J=11.88, 2.81 Hz), 4.22-4.31 (1H, m), 4.94-5.03 (2H, m), 5.68 (1H, m); ¹³C NMR (75 M Hz, CDCl₃) δ, 18.82, 21.70, 22.13, 22.37, 23.62, 24.18, 28.59, 31.34, 32.95, 34.76, 37.32, 43.73, 49.33, 51.09, 58.81, 65.39, 100.73, 115.21, 138.04, 209.56; HRMS (EI) Calc'd. for C₂₀H₃₄O₃: 322.2508. Found: 322.2497. [α]_(D) ²⁵=5.52° (c 4.79, CH₂Cl₂).

502.1 (Formula 502 where R⁸ is t-butyl)

To a solution of ketone 501 (35 mg, 0.109 mmol) in ether (0.6 mL) was added t-BuLi (1.6 M in pentane, 75 μL, 0.12 mmol) dropwise at −78° C. The mixture was stirred at rt for 30 min. and then quenched with sat. aq. NH₄Cl (2 mL). The mixture was allowed to warm to rt and was then extracted with EtOAc (3×5 mL). The combined organics were washed with brine, dried over Na₂SO₄, and concentrated in vacuo. Chromatography (10% EtOAc/hexane) afforded a major diastereomer A31a (17.5 mg, 42%) along with a minor diastereomer A31b. (7.4 mg, 18%) as colorless oils (structures not shown). A31a: IR (film): 2956, 1639, 1456 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ, 0.73 (1H, t, J=12.88 Hz), 0.89-0.94 (18H, m), 1.17-1.76 (14H, m), 2.00-2.09 (2H, m), 2.38-2.42 (1H, m), 2.73 (1H, d, J=13.73 Hz), 3.47 (1H, s), 3.79 (1H, dd, J=11.60, 5.06 Hz), 4.12 (1H, td, J=11.97, 2.44 Hz), 4.25-4.29 (1H, m), 4.91 (1H, d, J=10.17 Hz), 4.98 (1H, d, J=17.63 Hz), 5.77-5.84 (1H, m); ¹³C NMR (75 M Hz, CDCl₃) δ 19.07, 22.20, 22.25, 23.15, 23.45, 24.66, 25.43, 25.52, 28.98, 31.80, 34.36, 34.51, 34.60, 36.84, 37.54, 39.29, 39.63, 51.30, 58.92, 67.73, 79.0, 101.26, 114.31, 139.03; HRMS (EI) Calc'd. for C₂₄H₄₄O₃: 380.3290. Found: 380.3281. [α]_(D) ²⁵=5.89° (c 0.85, CDCl₃).

To a solution of major diastereomer A31a (332 mg, 0.872 mmol) in THF (5 mL) was added KHMDS (0.5M in toluene, 5.24 mL, 2.62 mmol) in an ice bath. TMSCl (284 mg, 2.62 mmol) was added and the mixture was stirred for 30 minutes. The mixture was quenched with sat. aq. NH₄Cl (5 mL) and extracted with EtOAc (3×5 mL). The combined organics were washed with brine (5 mL), dried over MgSO₄, and then concentrated in vacuo. Chromatography (5% EtOAc/hexane) providing 502.1 (395 mg, 100%) as a white solid. 502.1: m.p.=60.0° C.; IR (film) 2953, 1642, 1455 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) 6, 0.132 (6H, s), 0.134 (3H, s), 0.67-1.71 (33H, m), 1.95-1.99 (2H, m), 2.36-2.46 (1H, m), 2.71 (1H, app d, J=14.3 Hz), 3.74-3.79 (1H, m), 3.88-3.94 (1H, m), 4.07 (1H, td, J=11.75, 2.96 Hz), 4.95 (1H, d, J=10.41 Hz), 5.00 (1H, d, J=17.33 Hz), 5.74-5.85 (1H, m); ¹³C NMR (75 M Hz, CDCl₃) δ 2.9, 5.3, 18.9, 21.7, 22.2, 23.8, 24.3, 25.3, 26.2, 29.1, 34.2, 34.8, 35.0, 37.6, 39.2, 40.0, 51.3, 59.3, 65.6, 82.9, 100.7, 114.6, 138.7; HRMS (EI) Calc'd. for C₂₇H₅₂O₃Si: 452.3686. Found: 452.3670; [α]_(D) ²⁶=−11.33° (c 1.56, CDCl₃).

503.1 (Formula 503 where R⁸ is t-butyl)

To a solution of 502.1 (98 mg, 0.216 mmol) in CH₂Cl₂ (8 mL) and MeOH (0.5 mL) was bubbled O₃ at −78° C. until a blue color persists. Nitrogen gas was then used to purge the system and (EtO)₃P (54 mg, 0.324 mmol) was subsequently added. The mixture was stirred for 3 h and then slowly warmed to rt. Sat. aq. Na₂SO₃ (10 mL) was added and the mixture was extracted with CHCl₃ (3×10 mL). The combined organics were then washed with brine (10 mL), dried over MgSO₄ and concentrated in vacuo. Chromatography (0.5% EtOAc/hexane) afforded 503.1 (63 mg, 64.1%) as a colorless oil. 503.1: IR (film) 2941, 1715, 1454 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.134 (9H, s), 0.73 (1H, t, J=13.13 Hz), 0.9-1.81 (32H, m), 2.34-2.42 (3H, m), 2.72 (1H, d, J=12.29 Hz), 3.77 (1H, dd, J=11.59, 4.58 Hz), 3.92-3.96 (1H, m), 4.08 (1H, td, J=12.28, 2.01 Hz), 9.77 (1H, s); ¹³C NMR (100 M Hz, CDCl₃) 6, 3.39, 18.98, 19.29, 22.60, 24.23, 24.71, 26.75, 29.76, 34.41, 35.15, 37.77, 38.01, 39.66, 42.95, 45.26, 51.67, 59.64, 65.90, 83.08, 100.99, 202.26; HRMS (EI) Calc'd. for C₂₆H₅₀O₄Si: 454.3478. Found: 452.3475; [α]_(D) ²⁷=−6.33° (c 2.9, CH₂Cl₂).

To (−)-Ipc₂BOMe (108.8 mg, 0.34 mmol) (weighed in an inert atmosphere) was added diethyl ether (340 μL). The flask was cooled to −78° C. and allylmagnesium bromide (1.0 M, 310 μL, 0.31 mmol) was added. The precipitous mixture was stirred for 15 min. at −78° C. and then slowly warmed to rt and stirred for 1 hour. Diethyl ether (340 mL) was added and then a pre-cooled solution of aldehyde 503.1 (40.1 mg, 0.109 mmol) in diethyl ether (160 μL) was added dropwise. The suspension was stirred for 1 h, then the temperature was slowly raised to rt and 3N NaOH (240 μL), 30% hydrogen peroxide (100 μL) and diethyl ether (400 μL) was added. The biphasic mixture was refluxed for 1 h and then quenched with water (9 mL) and extracted with ethyl acetate (4×10 mL). The combined organics were washed with brine (10 mL), dried over sodium sulfate and the solvent was removed in vacuo. Chromatography on silica gel (12.5% EtOAc/hexane) afforded the crude homoallylic alcohol as a yellow oil. To this oil in methylene chloride (500 μL) was added imidazole (19 mg, 0.276 mmol) and TBSCl (21 mg, 0.138 mmol). The reaction was sealed and stirred for 14 h. The reaction was then quenched with a saturated solution of sodium bicarbonate (2 mL) and extracted with ethyl acetate (3×5 mL). The combined organics were washed with brine (5 mL), dried over sodium sulfate, filtered and the solvent was removed in vacuo. Filtration over silica gel (5% EtOAc/hexane) afforded a crude silyl ether A34 as a yellow oil (structure not shown). A34: IR (film) 2956, 1644, 1462 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.05 (6H, s), 0.05 (9H, s), 0.7-1.70 (42H, m), 2.21 (2H, d, J=6.48 Hz), 2.38-2.44 (2H, m), 2.72 (2H, d, J=13.36 Hz), 3.71 (1H, t, J=5.43 Hz), 3.77 (1H, dd, J=11.53, 3.59 Hz), 3.88-3.93 (1H, m), 4.07 (1H, td, J=1191, 3.06 Hz), 5.02 (1H, s), 5.06 (1H, d, J=3.66 Hz), 5.77-5.88 (1H, m); ¹³C NMR (100 M Hz, CDCl₃) δ −4.06, 3.39, 19.38, 21.48, 22.23, 22.72, 24.25, 24.78, 26.27, 26.69, 29.52, 34.58, 35.25, 38.06, 38.38, 38.50, 39.68, 42.02, 43.11, 59.68, 51.72, 65.98, 72.08, 83.20, 100.98, 117.06, 135.66; [α]_(D)=−4.18° (c 1.0, CDCl₃).

To crude silyl ether A34 in t-butanol (2.3 mL), water (1.4 mL) and pH 7 phosphate buffer (465 μL) was added NaIO₄ (79 mg, 0.368 mmol) followed by KMnO₄ (2.9 mg, 0.0184 mmol). The purple solution was stirred for 3 h and then water (3 mL) was added. This mixture was then extracted with ethyl acetate (4×5 mL) and the combined organics were washed with brine (5 mL), dried over sodium sulfate and filtered. The solvent was removed in vacuo and chromatography on silica gel (12.5% EtOAc, 0.1% acetic acid/hexanes) afforded acid 504.1 as well as the P—C3 diastereomeric alcohol. 504.1: IR (film): 3433, 2956, 1712, 1461 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.08 (3H, s), 0.09 (3H, s), 0.13 (6H, s), 0.70-1.70 (48H, m), 2.35-2.55 (3H, m), 2.72 (1H, d, J=12.74 Hz), 3.78 (1H, dd, J=11.34, 3.97 Hz,), 3.90-3.92 (1H, m), 4.75 (1H, dd, J=12.45, 11.97 Hz), 4.15 (1H, t, J=4.89 Hz); ¹³C NMR (100 M Hz, CDCl₃) δ −4.53, −4.10, 3.39, 18.32, 19.39, 21.29, 22.29, 22.66, 24.23, 24.75, 26.12, 26, 72, 29.64, 34.49, 35.19, 38.04, 38.38, 38.81, 39.71, 42.11, 43.08, 51.67, 59, 65, 65.93, 69.51, 83.10, 101.01, 176.69; [α]_(D) ²⁸=−8.19° (c 2.70, CDCl₃).

2D. 9-t-Butyl L4 Linker Synthon 508

To (−)-Ipc₂BOMe (108.8 mg, 0.34 mmol) (weighed in an inert atmosphere) was added diethyl ether (340 μL). The flask was cooled to −78° C. and allylmagnesium bromide (1.0 M, 310 μL, 0.31 mmol) was added. The precipitous mixture was stirred for 15 min. at −78° C. and then slowly warmed to rt and stirred for 1 hour. Diethyl ether (340 mL) was added and then a pre-cooled solution of an aldehyde 506 (0.109 mmol) (structure not shown) in diethyl ether (160 μL) was added dropwise. The suspension was stirred for 1 h, then the temperature was slowly raised to rt and 3N NaOH (240 μL), 30% hydrogen peroxide (100 μL) and diethyl ether (400 μL) was added. The biphasic mixture was refluxed for 1 h and then quenched with water (9 mL) and extracted with ethyl acetate (4×10 mL). The combined organics were washed with brine (10 mL), dried over sodium sulfate and the solvent was removed in vacuo. Chromatography on silica gel (12.5% EtOAc/hexane) afforded the crude homoallylic alcohol as a yellow oil. To this oil in methylene chloride (500 μL) was added imidazole (19 mg, 0.276 mmol) and TBSCl (21 mg, 0.138 mmol). The reaction was sealed and stirred for 14 h. The reaction was then quenched with a saturated solution of sodium bicarbonate (2 mL) and extracted with ethyl acetate (3×5 mL). The combined organics were washed with brine (5 mL), dried over sodium sulfate, filtered and the solvent was removed in vacuo. Filtration over silica gel (5% EtOAc/hexane) afforded a crude silyl ether as a yellow oil. To this oil in t-butanol (2.3 mL), water (1.4 mL) and pH7 phosphate buffer (465 μL) was added NaIO₄ (79 mg, 0.368 mmol) followed by KMnO₄ (2.9 mg, 0.0184 mmol). The purple solution was stirred for 3 h and then water (3 mL) was added. This mixture was then extracted with ethyl acetate (4×5 mL) and the combined organics were washed with brine (5 mL), dried over sodium sulfate and filtered. The solvent was removed in vacuo and chromatography on silica gel (12.5% EtOAc, 0.1% acetic acid/hexanes) afforded acid 508.1 (34 mg, 56%) as a colorless oil. 508.1: R_(f) (15% ethyl acetate/hexanes)=0.17; IR (film) 2700-3300, 2954, 2869, 1713, 1107, 837, 776 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.08 (6H, s), 0.69 (1H, t, J=13.1 Hz), 0.76-0.90 (27H, m), 1.17-1.25 (3H, m), 1.39-1.61 (6H, m), 1.70-1.82 (3H, m), 2.40 (1H, dsept, J=7.2, 0.9 Hz), 2.58 (1H, dd, J=13.1, 5.6 Hz), 2.68 (1H, br d, J=12.6 Hz), 2.91 (1H, t, J=4.8 Hz), 3.41 (1H, dd, J=5.9, 2.3 Hz), 3.63-3.77 (2H, m), 3.82 (1H, dd, J=11.7, 4.2 Hz), 4.066 (1H, td, J=12.0, 2.7 Hz), 4.23-4.29 (1H, m), 9.20-9.40 (1H, br s); ¹³C NMR (100 M Hz, C₆D₆) δ −5.1, −4.8, 17.8, 18.9, 21.9, 22.1, 23.7, 24.3, 25.6, 26.0, 29.1, 31.8, 34.9, 35.7, 37.3, 38.7, 41.8, 51.3, 59.2, 66.6, 66.9, 67.2, 84.5, 174.7; HRMS (FAB) Calc'd. for C₃₀H₅₈O₆Si: 542.4002. Found: 542.4005; [α]_(D) ²²=−5.88° (c 1.67, CH₂Cl₂).

2E. Linker Synthon 507

To (−)-Ipc₂BOMe (108.8 mg, 0.34 mmol) (weighed in an inert atmosphere) was added diethyl ether (340 μL). The flask was cooled to −78° C. and allylmagnesium bromide (1.0 M, 310 μL, 0.31 mmol) was added. The precipitous mixture was stirred for 15 min. at −78° C. and then slowly warmed to rt and stirred for 1 hour. Diethyl ether (340 mL) was added and then a pre-cooled solution of an aldehyde 506 (0.109 mmol) (structure not shown) in diethyl ether (160 μL) was added dropwise. The suspension was stirred for 1 h, then the temperature was slowly raised to rt and 3N NaOH (240 μL), 30% hydrogen peroxide (100 μL) and diethyl ether (400 μL) was added. The biphasic mixture was refluxed for 1 h and then quenched with water (9 mL) and extracted with ethyl acetate (4×10 mL). The combined organics were washed with brine (10 mL), dried over sodium sulfate and the solvent was removed in vacuo. Chromatography on silica gel (12.5% EtOAc/hexane) afforded the crude homoallylic alcohol A40 (structure not shown) as a yellow oil. R_(f) (35% ethyl acetate/hexanes)=0.65; IR (film) 3455, 2950, 2868, 1456, 1373, 1308, 1265, 1110, 997 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.68 (1H, t, J=13.5 Hz), 0.83-0.92 (10H, m), 1.39-1.52 (5H, m), 1.57-1.72 (3H, m), 1.71 (1H, d, J=4.9 Hz), 2.24 (1H, t, J=4.9 Hz), 2.35-2.42 (2H, m), 2.70 (3H, br d, J=12.4 Hz), 3.46-3.60 (3H, m), 3.62-3.70 (1H, m), 3.77-3.95 (3H, m), 4.11 (1H, dd, J=11.9, 2.7 Hz), 5.07 (2H, dd, J=9.5, 1.9 Hz), 5.70-5.91 (1H, m); ¹³C NMR (100 M Hz, CDCl₃) δ 13.9, 16.4, 17.5, 21.1, 22.0, 23.1, 29.1, 30.7, 31.1, 31.8, 33.7, 37.3, 46.1, 51.1, 56.9, 64.7, 65.0, 66.8, 113.3, 129.3; [α]_(D) ²⁰=2.5° (c 0.8, CDCl₃).

To A40 in methylene chloride (500 μL) was added imidazole (19 mg, 0.276 mmol) and TBSCl (21 mg, 0.138 mmol). The reaction was sealed and stirred for 14 h. The reaction was then quenched with a saturated solution of sodium bicarbonate (2 mL) and extracted with ethyl acetate (3×5 mL). The combined organics were washed with brine (5 mL), dried over sodium sulfate, filtered and the solvent was removed in vacuo. Filtration over silica gel (5% EtOAc/hexane) afforded a crude silyl ether A41 (structure not shown) as a yellow oil. R_(f) (35% ethyl acetate/hexanes)=0.65; IR (film): 2953, 2864, 1641, 1472, 1372, 1255, 1108, 830 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.05 (3H, s), 0.05 (3H, s), 0.67 (1H, t, J=13.5 Hz), 0.81-0.96 (18H, m), 1.11-1.20 (1H, m), 1.35-1.47 (4H, m), 1.52-1.71 (5H, m), 2.22 (1H, br s), 2.35-2.42 (2H, m), 2.70 (3H, br d, J=12.2 Hz), 3.38-3.54 (4H, m), 3.78-3.97 (3H, m), 4.10 (1H, dd, J=11.9, 2.8 Hz), 5.05 (2H, dd, J=9.4, 1.8 Hz), 5.69-5.91 (1H, m); ¹³C NMR (100 M Hz, CDCl₃) δ −9.5, −9.1, 13.3, 14.1, 17.1, 17.5, 18.9, 19.5, 21.1, 24.1, 27.0, 30.2, 31.8, 32.0, 32.6, 37.5, 46.4, 54.4, 59.5, 61.9, 62.8, 64.1, 95.6, 112.0, 130.0; [α]_(D) ²⁰=16.2° (c 1.0, CH₂Cl₂).

To A41 in t-butanol (2.3 mL), water (1.4 mL) and pH 7 phosphate buffer (465 μL) was added NaIO₄ (79 mg, 0.368 mmol) followed by KMnO₄ (2.9 mg, 0.0184 mmol). The purple solution was stirred for 3 h and then water (3 mL) was added. This mixture was then extracted with ethyl acetate (4×5 mL) and the combined organics were washed with brine (5 mL), dried over sodium sulfate and filtered. The solvent was removed in vacuo and chromatography on silica gel (12.5% EtOAc, 0.1% acetic acid/hexanes) afforded acid 507. IR (film): 2700-3300, 2952, 2866, 1738, 1471, 1373, 1307, 1146, 1103, 837, cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.06 (3H, s), 0.07 (3H, s), 0.67 (1H, app t, J=13.1 Hz), 0.81-0.89 (19H, m), 1.14-1.20 (1H, m), 1.35-1.47 (5H, m), 1.50-1.82 (4H, m), 2.34-2.41 (1H, m), 2.45-2.53 (2H, m), 2.69 (3H, br d, J=13.7 Hz), 3.44-3.51 (4H, m), 3.80 (1H, dd, J=11.5, 3.9 Hz), 3.87-3.95 (1H, m), 4.02-4.09 (1H, m), 4.22-4.29 (1H, m); ¹³C NMR (100 M Hz, CDCl₃) δ −9.6, 13.1, 14.1, 17.1, 17.5, 18.9, 19.5, 20.9, 24.2, 26.9, 30.1, 32.0, 32.3, 32.6, 37.7, 46.4, 54.3, 59.6, 61.9, 62.1, 95.7, 171.5; HRMS (FAB) Calc'd. for C₂₆H₅₀O₆Si: 486.3379. Found: 486.3377; [α]_(D) ²⁰=9.4° (c 1.3, CH₂Cl₂).

2F. Ether Diester Linker Synthon 606

The following procedure, referred to as the “general isolation procedure”, was used to purify various reaction products below. The reaction mixture is quenched by dropwise addition of saturated aqueous ammonium chloride, and the resultant mixture is allowed to partition between solvent and brine or water. The aqueous layer is extracted with 1 to 3 portions of ethyl acetate. The combined organic extracts are dried over sodium sulfate and concentrated in vacuo.

Formula 602

To a solution of 3-(p-methoxybenzyloxy)propyl allyl ether 601 (1.0 g, 4.3 mmol) in THF (10 mL) was added 9-BBN (20.6 mL of 0.5 M solution in THF, 10.3 mmol), and the mixture was stirred for 2 h at rt. Hydrogen peroxide (30%, 10 mL) and sodium hydroxide (15%, 10 mL) were added and the mixture was stirred for 3 h. The general isolation procedure afforded crude product which was purified further by silica gel chromatography to give the expected purified alcohol A51 (870 mg, 75%) (structure not shown). R_(f)=0.25 (50% ethyl acetate in hexane); IR (neat)=3423, 2934, 2864, 1612, 1513, 1248, 1098 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 7.25 (d, J=8.7 Hz, 2H), 6.87 (d, J=8.7 Hz, 2H), 4.43 (s, 2H), 3.80 (s, 3H), 3.75 (q, J=5.7 Hz, 2H), 3.60 (t, J=5.7 Hz, 2H), 3.52 (m, 4H), 2.46 (br t, 1H), 1.84 (m, 4H); ¹³C NMR (75 M Hz, CDCl₃) δ 129.36, 113.83, 72.63, 70.34, 68.33, 66.89, 62.20, 55.23, 31.88, 29.96.

To a solution of DMSO (1.38 mL, 19.5 mmol) in methylene chloride (5 mL) at −78° C. was added oxalyl chloride (850:L, 9.74 mmol) dropwise. After 5 min, alcohol A51 from the preceding step (870 mg, 6.49 mmol) in methylene chloride (2 mL) was added and the mixture was stirred for another 20 min. TEA (triethylamine, 4.31 mL, 32.5 mmol) was added. After 10 min, the mixture was warmed to rt. The standard isolation procedure afforded the expected aldehyde 602 (760 mg, 89%). R_(f)=0.40 (33% ethyl acetate in hexane); IR (neat)=2863, 1724, 1612, 1513, 1248, 1098 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 9.75 (s, 1H), 7.25 (d, J=9.0 Hz, 2H), 6.87 (d, J=9.0 Hz, 2H), 4.42 (s, 2H), 3.80 (s, 3H), 3.74 (t, J=6.0 Hz, 2H), 3.51 (m, 4H), 2.62 (m, 2H), 1.84 (quint, J=6.3 Hz, 2H); ¹³C NMR (75 M Hz, CDCl₃) δ 201.49, 159.26, 130.65, 129.31, 113.78, 72.57, 68.08, 66.69, 64.40, 55.19, 43.75, 29.86.

Formula 603

(−)-Ipc₂BOMe (1.91 g, 6.04 mmol) in ether (4 mL) was treated with allylmagnesium bromide (4.83 mL, 4.83 mmol) at rt. After 30 min, the mixture was cooled to −78° C. The aldehyde 602 (370 mg, 2.76 mmol) in ether (1.5 mL) was added and stirred for 2 h at −78° C. 15% NaOH (1.5 mL) and 30% hydrogen peroxide (1.5 mL) were added and the mixture was warmed to rt. After 2 h, the mixture was diluted with ethyl acetate and washed with brine. Column chromatography yielded the expected alcohol A53 (383 mg, 80%) (structure not shown). Alcohol A53 (150 mg, 0.852 mmol) in methylene chloride (4 mL) was treated with TBSCl (168 mg, 1.11 mmol) and imidazole (116 mg, 1.7 mmol) at rt. After overnight, the reaction was worked up by the standard procedure. Column chromatography afforded expected TBS alkene ether 603 (195 mg, 79%). 603: R_(f)=0.50 (10% ethyl acetate in hexane); [α]²⁵ _(D)=13.6° (c 1.08, CH₂Cl₂); IR (neat)=2952, 2856, 1717, 1613, 1513, 1471, 1362, 1248, 1110 cm⁻¹; ¹HNMR (400 M Hz, CDCl₃) δ 7.26 (d, J=8.4 Hz, 2H), 6.88 (d, J=8.4 Hz, 2H), 5.81 (m, 1H), 5.04 (m, 2H), 4.43 (s, 2H), 3.86 (s, 3H), 3.49 (m, 7H), 2.23 (m, 2H), 1.87 (m, 2H), 1.69 (m, 2H), 0.89 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H); ¹³C NMR (100 M Hz, CDCl₃) δ 159.09, 134.96, 130.60, 116.93, 113.71, 72.59, 68.91, 67.80, 67.48, 67.18, 55.23, 42.28, 36.62, 30.16, 25.85, 18.08, −4.39, −4.76.

Formula 605

Alkene ether 603 (195 mg, 0.672 mmol) in tert-butanol-water (pH 7) (1:1, 4 mL) was treated with KMnO₄ (11 mg, 0.0672 mmol) and sodium periodate (548 mg, 2.56 mmol) in water (0.5 mL). After 30 min, the reaction was quenched with sodium thiosulfate. The mixture was diluted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and concentrated to give a crude acid 604. The crude acid in methylene chloride (3 mL) was treated with SEMCl (2-(trimethylsilyl)ethoxymethyl chloride, 301:L, 1.7 mmol) and TEA (452:L, 3.4 mmol) at rt and stirred for 2 h. The reaction was worked up by the standard procedure to afford the expected SEM ester 605.

SEM ester 605 in wet methylene chloride (3 mL) was treated with DDQ (291 mg, 1.28 mmol). After 1 h, the mixture was directly purified by silica gel chromatography to give the alcohol product 606 (86 mg, 29% for 3 steps). 606: Rf=0.25 (25% ethyl acetate in hexane); [α]²⁵ _(D)=0.4° (c 0.82, CH₂Cl²); IR (neat)=3458, 2955, 1741, 1472, 1378, 1250, 1116 cm¹; ¹H NMR (400 M Hz, CDCl₃) δ 5.25 (q, J=2.8 Hz, 2H), 4.25 (quint, J=6.4 Hz, 2H), 3.71 (m, 4H), 3.52 (m, 4H), 2.50 (d, J=6.0 Hz, 2H), 2.44 (br, 1H), 1.79 (m, 4H), 0.94 (t, J=8.4 Hz, 2H), 0.85 (s, 9H), 0.05 (s, 3H), 0.03 (s, 3H), 0.00 (s, 9H); ¹³C NMR (100 M Hz, CDCl₃) δ 171.04, 88.97, 69.88, 67.88, 67.25, 66.45, 61.81, 42.84, 37.12, 32.02, 25.71, 17.97, 17.91, −1.48, −4.81, −4.86

2G. C5 Ester-Open C7 Linker Synthon 513

To menthone aldehyde 402 (400 mg, 1.56 mmol) from Example 2A in DMF (12 mL) was added prenyl bromide (396 mg, 1.56 mmol) followed by indium powder (359 mg, 3.13 mmol) at rt. After 30 min., the reaction was diluted with EtOAc (20 mL) and saturated aqueous ammonium chloride (15 mL). The layers were separated and the aqueous layer was re-extracted with EtOAc (3×10 mL). The combined organics were washed with brine (15 mL) and dried over sodium sulfate. The solvent was removed in vacuo and chromatography (7.5% EtOAc/pentane) to afford 509a (105 mg, 21%) and 509b (321 mg, 63%).

Undesired isomer 509a can be recycled by the following procedure: To 509a (320.8 mg, 0.98 mmol) in methylene chloride (3 mL) was added Dess-Martin Periodinane (707 mg, 1.67 mmol) and stirred for 1 h at room temperature. The reaction was diluted with methylene chloride (3 mL), saturated sodium bicarbonate (3 mL) and sodium thiosulfate (3 mL) and stirred for 1 h. The layers were separated and the aqueous layer was re-extracted with EtOAc (4×5 mL). The combined organics were dried over sodium sulfate and the solvent was removed in vacuo. The crude ketone was dissolved in MeOH (17.3 mL) and CeCl₃.7H₂O (1.83 g, 4.9 mmol) was added and stirred for 5 min. The solution was cooled to −50° C. and NaBH₄ (74.5 mg, 19.6 mmol) was added. The reaction was stirred for 30 min. and then poured into a separatory funnel containing EtOAc (30 mL), water (18 mL) and brine (30 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3×10 mL). The combined organics were dried over sodium sulfate and the solvent was removed in vacuo. Chromatography (7.5%-20% EtOAc/pentane) provided 509b (177 mg, 53%) and 509a (56 mg, 18%). 509a: R_(f) (7.5% EtOAc/pentane)=0.186; IR (film): 3496.8, 2953.9, 2869.6, 1458.0, 1374.4, 1308.3, 1266.5, 1157.8, 1130.7, 1102.0, 977.6, 912.2 cm⁻¹; ¹H-NMR (300 M Hz, CDCl₃) δ 0.68 (1H, t, J=12.9 Hz), 0.85-0.89 (9H, m), 0.98 (3H, s), 0.99 (3H, s), 1.14-1.76 (10H, m), 2.38 (1H, sept, J=6.9 Hz), 2.73 (1H, br. d, J=13.5 Hz), 3.59 (1H, br. d, J=9.9 Hz), 3.78 (1H, dd, J=11.7, 5.4 Hz), 4.02 (1H, d, J=11.4), 4.11 (1H, d, J=13.2), 5.00-5.09 (2H, m), 5.81 (1H, dd, J=17.6, 11.0 Hz); ¹³C-NMR (75 MHz, CDCl₃) δ 18.8, 21.7, 22.0, 22.4, 23.1, 23.8, 24.3, 29.1, 31.9, 34.9, 37.4, 38.5, 41.2, 51.2, 59.1, 64.5, 73.5, 100.4, 113.2, 145.4; HRMS calc'd for C₂₀H₃₆O₃: 324.2664. found: 324.2664; [α]^(24.0) _(d): +19.36° (c 8.16, CH₂Cl₂). 509b: R_(f) (7.5% EtOAc/pentane)=0.37; IR (film): 3520.4, 2953.6, 2870.2, 1455.8, 1375.6, 1309.4, 1266.0, 1130.3, 1090.0, 973.3, 914.4 cm⁻¹; ¹H-NMR (300 M Hz, CDCl₃) δ 0.73 (1H, t, J=13 Hz), 0.83-0.92 (9H, m), 1.00 (3H, s), 1.01 (3H, s), 1.18-1.77 (10H, m), 2.39 (1H, sept, J=7 Hz), 2.73 (1H, br. d, J=13.5 Hz), 3.53 (1H, d, J=9.9 Hz), 3.57 (1H, s), 3.79 (1H, dd, J=11.7, 4.8 Hz), 4.00-4.15 (2H, m), 4.96-5.02 (2H, m), 5.87 (1H, dd, J=17.1, 11.1 Hz); ¹³C-NMR (75 M Hz, CDCl₃) δ 18.9, 21.9, 22.2, 22.3, 23.3, 23.8, 24.2, 28.9, 31.8, 34.6, 37.3, 38.0, 41.1, 50.9, 58.8, 70.3, 78.9, 101.0, 111.9, 145.7; HRMS calc'd for C₂₀H₃₆O₃: 324.2664. found: 324.2665; [α]^(25.1) _(d): −1.94° (c 9.75, CH₂Cl₂).

Formula 510

To a solution of 509b (282 mg, 0.87 mmol) in MeOH (3.3 mL) and methylene chloride (13 mL) at −78° C. was bubbled ozone for 5 min. or until a blue color persisted. The solution was then purged with nitrogen for 5 min. NaBH₄ (138 mg, 3.65 mmol) was then added and stirred for 1 h at −78° C., and 2 h at 0° C. Saturated ammonium chloride (15 mL) and water (5 mL) were then poured into the reaction and the layers were separated. The aqueous layer was extracted with methylene chloride (4×5 mL). The combined organics were dried over sodium sulfate and the solvent was removed in vacuo, yielding crude diol 510: R_(f) (20% EtOAc/pentane)=0.195; IR (film): 3452.3, 2954.1, 2871.3, 1455.5, 1383.4, 1308.9, 1266.9, 1131.4, 1047.2, 972.6 cm⁻¹; ¹H-NMR (300 M Hz, CDCl₃) δ 0.74 (1H, t, J=13 Hz), 0.85-0.89 (12H, m), 0.92 (3H, d, J=6.6 Hz), 1.12-1.25 (2H, m), 1.39-1.78 (7H, m), 2.39 (1H, sept, J=6.9 Hz), 2.73 (1H, br. d, J=13.5 Hz), 3.48 (3H, m), 3.70-3.73 (1H, m), 3.80 (1H, dd, J=11.4, 5.1 Hz), 4.08-4.15 (3H, m); ¹³C-NMR (75 M Hz, CDCl₃) δ 18.8, 19.0, 22.2, 22.3, 22.5, 23.7, 24.2, 29.0, 31.8, 34.5, 37.3, 37.6, 38.0, 50.9, 58.8, 70.6, 72.0, 80.5, 101.0; HRMS calc'd for C₁₉H₃₆O₄: 328.2614. found: 328.2619; [α]^(26.0) _(d): −0.78° (c 5.48, CH₂Cl₂).

Formula 511

To crude diol 510 was added pyridine (316 μL, 3.91 mmol) and chloroacetic anhydride (229 mg, 1.3 mmol) at −78° C. and stirred for 2 hr. Saturated aqueous sodium bicarbonate (10 mL) was added and the layers were separated. The aqueous layer was extracted with methylene chloride (3×5 mL). The combined organics were dried over sodium sulfate and the solvent was removed in vacuo. Chromatography (10%-20%-30% EtOAc/pentane) provided residual starting material A61 (89.8 mg, 31%) and chloroacetate ester 511 (152.5 mg, 44%). 511: R_(f) (20% EtOAc/pentane)=0.56; IR (film): 3515.2, 2954.4, 2871.9, 1738.5, 1455.8, 1371.6, 1308.7, 1132.8, 972.7 cm⁻¹; ¹H-NMR (300 M Hz, CDCl₃) δ 0.75 (1H, t, J=13.2 Hz), 0.87 (3H, d, J=2.7 Hz), 0.89 (3H, d, J=3 Hz), 0.92-0.93 (10H, m), 1.19-1.26 (2H, m), 1.39-1.79 (7H, m), 2.40 (1H, quin, J=6.9 Hz), 2.74 (1H, br. d, J=13.2 Hz), 3.65-3.66 (2H, m), 3.81 (1H, dd, J=11.6, 4.7 Hz), 4.00-4.17 (4H, m), 4.07 (2H, s); ¹³C-NMR (75 M Hz, CDCl₃) δ 18.9, 19.3, 21.4, 22.2, 22.3, 23.8, 24.2, 29.0, 31.8, 34.5, 37.3, 37.4, 38.2, 40.9, 50.9, 58.8, 70.5, 71.6, 76.1, 101.0, 167.3; HRMS calc'd for C₂₁H₃₇ClO₅: 404.2330. found: 404.2329; [α]^(24.3) _(d): +4.95° (c 3.43, CH₂Cl₂).

Formula 512

To acid 28 (structure not shown)-1-(1′-naphthyl)ethyl 3-[(tert-butyldimethylsilyl)oxy]pentanedioate, 309 mg, 0.744 mmol) in toluene (6 mL) was added triethylamine (263 μL, 1.98 mmol) followed by the Yamaguchi reagent (124 μL, 0.79 mmol) and stirred for 2 hr at room temperature. DMAP (303 mg, 2.50 mmol) was then added followed by 511 (190 mg, 0.469 mmol) and the mixture was stirred for 45 min. The reaction was then diluted with EtOAc (5 mL) and saturated aqueous sodium bicarbonate (10 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3×5 mL). The combined organics were washed with saturated aqueous ammonium chloride (10 mL), the layers were separated and the aqueous layer was extracted with EtOAc (2×5 mL). The combined organics were dried over sodium sulfate and the solvent was removed in vacuo. Chromatography (5%-10% EtOAc/pentane) provided 512 (311.9 mg, 83%): R_(f) (10% EtOAc/pentane)=0.46; IR (film): 2953.8, 2868.2, 1738.1, 1471.9, 1373.6, 1307.9, 1258.5, 1160.3, 1108.7, 1069.3, 977.5, 837.0, 777.9 cm⁻¹; ¹H-NMR (500 M Hz, CDCl₃) δ 0.04 (3H, s), 0.12 (3H, s), 0.61 (1H, t, J=13.2 Hz), 0.79 (9H, s), 0.82-0.92 (9H, m), 0.94 (3H, s), 0.95 (3H, s), 1.12-1.15 (1H, m), 1.35-1.47 (1H, m), 1.56-1.65 (4H, m), 1.69 (3H, d, J=6.5 Hz), 1.72-1.79 (1H, m), 2.37 (1H, quin, J=6.9 Hz), 2.56-2.63 (4H, m), 2.68 (1H, dd, J=15.3, 5.3 Hz), 3.64-3.68 (1H, m), 3.76 (1H, dd, J=11.8, 4.8 Hz), 3.86 (1H, d, J=11 Hz), 3.97 (1H, td, J=12.6, 1.8 Hz), 4.01 (1H, d, J=11 Hz), 4.05 (2H, s), 4.51 (1H, quin, J=6 Hz), 4.98 (1H, dd, J=9.5, 1.0 Hz), 6.64 (1H, q, J=6.5 Hz), 7.42-7.53 (3H, m), 7.57 (1H, d, J=7.0 Hz), 7.78 (1H, d, J=8.0 Hz), 7.85 (1H, d, J=8.5 Hz), 8.06 (1H, d, J=8.5 Hz)); ¹³C-NMR (125 M Hz, CDCl₃) δ −5.1, −4.8, 14.2, 17.8, 19.0, 20.3, 21.4, 21.7, 21.8, 22.3, 23.7, 24.3, 25.6, 28.8, 31.0, 34.9, 37.2, 37.3, 38.1, 40.7, 41.8, 42.1, 51.1, 58.9, 65.6, 65.7, 69.7, 70.9, 73.0, 100.5, 123.1, 123.2, 125.3, 125.6, 126.2, 128.4, 128.8, 130.1, 133.8, 137.3, 167.1, 170.1, 170.4; HRMS calc'd for C₄₄H₆₇ClO₉Si (+1Na): 825.4149. found: 825.4141; [α]^(25.5) _(d): −2.17° (c 8.42, CH₂Cl₂).

Formula 513

To 512 (156 mg, 0.194 mmol) in EtOAc (4.4 mL) at room temperature was added Pd(OH)₂/C (75 mg). The black slurry was stirred and the flask was evacuated and refilled with hydrogen (5 times). After 5.5 h under 1 atm. of hydrogen, the reaction was poured directly onto a silica column pre-packed with pentane and eluted (20% EtOAc+1% AcOH/pentane) to provide 119 mg of linker synthon 513 in 95% yield. 513: R_(f) (20% EtOAc+1% AcOH/pentane)=0.27; IR (film): 2800.0-3422.4, 2954.1, 2866.6, 1738.3, 1714.1, 1473.1, 1375.8, 1308.0, 1258.4, 1159.6, 1107.3, 977.0, 837.3, 778.8 cm⁻¹; ¹H-NMR (300 M Hz, CDCl₃) δ 0.08 (3H, s), 0.09 (3H, s), 0.66 (1H, t, J=13.0 Hz), 0.85-0.92 (19H, m), 0.95 (3H, s), 0.97 (3H, s), 1.06-1.25 (1H, m), 1.33-1.50 (4H, m), 1.58-1.81 (4H, m), 2.37 (1H, dsept, J=6.6, 1.2 Hz), 2.53 (1H, dd, J=15.3, 6.9 Hz), 2.58-2.60 (3H, m), 2.66 (1H, dd, J=15.0, 4.8 Hz), 3.64-3.68 (1H, m), 3.79 (1H, dd, J=11.7, 4.2 Hz), 3.86 (1H, d, J=11.1 Hz), 3.98-4.03 (1H, m), 4.00 (1H, d, J=11.1 Hz), 4.06 (2H, s), 4.49 (1H, quin, J=5.9 Hz), 4.98 (1H, dd, J=9.6, 1.8 Hz)); ¹³C-NMR (75 M Hz, CDCl₃) δ −5.1, −4.8, 17.9, 19.0, 20.3, 21.4, 21.7, 22.3, 23.7, 24.3, 25.7, 28.8, 31.1, 34.9, 37.3, 37.4, 38.2, 40.7, 41.9, 42.0, 51.1, 58.9, 65.7, 65.7, 70.9, 73.3, 100.6, 167.2, 170.2, 176.4; HRMS calc'd for C₃₂H₅₇ClO₉Si (+1Na): 671.3348. found: 671.3358; [α]^(27.4) _(d): −20.46° (c 7.46, CH₂Cl₂).

Example 3 Exemplary Bryostatin Analogues 3A. Formula II—C26 Des-Methyl Bryostatin Analogue (702)

To di-benzyl ether 111.1 (Example IC, 503 mg, 0.169 mmol) in EtOAc (12.3 mL) at room temperature is added Pd(OH)₂/C (82 mg). The black slurry was stirred vigorously and the flask was evacuated and refilled 4 times with hydrogen (1 atm). After 1 h, the reaction was poured directly onto a silica column and eluted (50% EtOAc/pentane to 100% EtOAc) to provide 201.1 (240.2 mg, 65%) as a colorless oil.

201.1: R_(f) (50% EtOAc/pentane)=0.36; IR (film)=3424, 2955, 2930, 2857, 1748, 1722, 1156, 1081, 837, 776 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.00 (6H, s), 0.84-0.93 (12H, m), 0.96 (3H, s), 0.98 (3H, s), 1.20 (3H, d, J=6.0 Hz), 1.15-1.38 (10H, m), 1.62 (1H, t, J=7.1 Hz), 1.71 (1H, t, J=5.7 Hz), 2.25-2.45 (2H, m), 2.54 (1H, s), 2.89 (1H, d, J=4.2 Hz), 3.34-3.48 (1H, m), 3.36 (3H, s), 3.52 (2H, dd, J=15.9, 9.6 Hz), 3.58-3.76 (3H, m), 3.67 (3H, s), 4.14-4.26 (1H, m), 5.55 (1H, s), 5.89 (1H, s); ¹³C NMR (75 M Hz, CDCl₃) δ −5.5, 14.0, 18.4, 19.4, 20.7, 22.5, 24.6, 25.9, 28.8, 29.0, 31.6, 32.4, 34.4, 39.0, 46.6, 51.1, 51.3, 67.5, 68.4, 70.9, 71.9, 72.3, 103.1, 116.9, 152.4, 166.5, 171.8; [α]^(23.9) _(D)=−2.24° (c=6.53, CH₂Cl₂).

To diol 201.1 (26.8 mg, 0.045 mmol) in benzene (1.2 mL) under nitrogen at 0° C. was added triethylamine (30 μL, 0.227 mmol) followed by lead tetraacetate (50 mg, 0.113 mmol). The resulting suspension was stirred at 0° C. for 20 min. and was then quenched with an aqueous solution of saturated ammonium chloride (5 mL) and extracted with ethyl acetate (3×5 mL). The combined organic layers were dried over sodium sulfate, the solution was decanted and then the solvent was removed in vacuo to provide the crude aldehyde (structure not shown) which was taken immediately to the next step.

To the crude aldehyde (34 mg, 0.061 mmol) in THF (1.4 mL) under nitrogen at 0° C. was added a 0.5M solution of the Tebbe reagent in toluene (122 μL, 0.061 mmol) dropwise. The reddish-black slurry was stirred at 0° C. for 15 min. and was then quenched with a saturated aqueous solution of sodium bicarbonate (5 mL). The biphasic mixture was extracted with ethyl acetate (3×5 mL). The combined organic layers were dried over sodium sulfate, the solution was decanted and then the solvent was removed in vacuo. Chromatography (5% EtOAc/pentane) provides olefin 202.1 (19 mg, 56%—2 steps) as a colorless oil.

202.1: R_(f) (10% EtOAc/pentane)=0.62; IR (film)=2955, 2930, 2857, 1747, 1722, 1667, 1155, 1082, 837, 775 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.01 (6H, s), 0.80-0.90 (12H, m), 0.96 (3H, s), 1.00 (3H, s), 1.20-1.38 (10H, m), 1.56-1.72 (1H, m), 2.24-2.46 (4H, m), 3.43 (1H, d, J=15.9 Hz), 3.30 (3H, s), 3.54 (2H, dd, J=18.9, 9.3 Hz), 3.68 (3H, s), 3.87-3.91 (1H, m), 5.09-5.16 (2H, m), 5.53 (1H, s), 5.80-5.98 (1H, m), 5.88 (1H, s); ¹³C NMR (75 M Hz, CDCl₃) δ −5.4, 14.0, 18.4, 20.5, 20.6, 22.6, 24.7, 25.9, 28.9, 29.0, 31.6, 32.1, 34.5, 40.0, 47.0, 51.1, 67.3, 71.0, 72.1, 103.0, 116.6, 117.7, 133.8, 153.1, 166.6, 171.9; [α]^(22.0) _(D)=−7.91° (c=1.91, CH₂Cl₂).

To a solution of silyl ether 202.1 (101.7 mg, 0.1833 mmol) and pyridine (267 μL, 3.30 mmol) in THF (1.53 mL) in a polypropylene vial was added 70% HF/pyridine complex (104.8 μL, 3.67 mmol) at room temperature. The solution was stirred for 18 hours and was then quenched with a saturated aqueous solution of sodium bicarbonate (5 mL). The biphasic mixture was extracted with ethyl acetate (4×5 mL). The combined organic layers were dried over sodium sulfate, the solution was decanted, and the solvent was removed in vacuo to afford the corresponding de-silylated alcohol (structure not shown) as a pale yellow oil which was used immediately in the next step.

The crude alcohol was dissolved in CH₂Cl₂ (2 mL) and treated with a single portion of 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (Dess-Martin periodinane, 117 mg, 0.275 mmol) at room temperature. The mixture was stirred for 45 min and quenched with saturated aqueous NaHCO₃/Na₂S₂O₃ (2 mL). The two phase system was vigorously stirred until the organic layer has cleared (90 min). The layers were then separated and the aqueous phase was extracted with CH₂Cl₂ (4×5 mL). The combined organic layers were dried over sodium sulfate, the solution was decanted and then the solvent was removed in vacuo. Chromatography on silica gel (7.5% EtOAc/hexanes) provides aldehyde 203.1 (43.7 mg, 54%—2 steps) as a colorless oil.

203.1: R_(f) (15% EtOAc/pentane)=0.58; IR (film)=2930, 2857, 1750, 1723, 1668, 1160, 1048 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.86 (3H, t, J=6.3 Hz, octanoate Me), 1.00 (3H, s, C18 Me), 1.16 (3H, s, C18 Me), 1.18-1.31 (10H, m), 1.46-1.60 (2H, m), 2.18 (2H, t, J=7.4 Hz), 2.43 (2H, t, J=6.6 Hz), 3.39 (3H, s), 3.66 (1H, d, J=18.9 Hz), 3.69 (3H, s), 3.74-3.84 (1H, m), 5.13-5.20 (2H, m), 5.85-6.00 (1H, m), 5.96 (1H, s), 9.71 (1H, s); ¹³C NMR (75 M Hz, CDCl₃) δ 14.1, 16.3, 19.1, 22.6, 24.3, 28.9, 30.4, 31.6, 38.9, 40.0, 51.2, 51.4, 54.0, 71.5, 71.9, 102.2, 118.2, 119.8, 133.4, 150.3, 166.4, 171.7, 202.4; [α]^(23.6) _(D)=−6.07° (c=2.23, CH₂Cl₂).

A dihydroxylating stock solution was generated by dissolving (DHQD)₂AQN (3.6 mg, 0.00425 mmol), K₃Fe(CN)₆ (425 mg, 1.275 mmol), K₂CO₃ (175 mg, 1.275 mmol) and K₂OsO₂ (OH)₄ (0.65 mg, 0.00175 mmol) in t-BuOH (2.1 mL) and water (2.1 mL). The resulting solution was stirred at room temperature for 3 h. 504 μL of this stock solution was added to olefin 203.1 (7.4 mg, 0.017 mmol) pre-dissolved in t-BuOH (200 μL) and water (200 μL) under nitrogen at 0° C. The resulting solution was stirred at 0-5° C. for 2 days. Water (2 mL) was then added and the biphasic mixture was then extracted with EtOAc (4×5 mL). The combined organic layers were dried over sodium sulfate, the solution was decanted and then the solvent was removed in vacuo. Chromatography (90% EtOAc/pentane to 100% EtOAc) provides diol 204.1 (5.6 mg, 70%) as an approximately 2.2:1 (β:α) mixture of diastereomers as a colorless oil.

204.1: R_(f) (90% EtOAc/pentane)=0.36; IR (film)=3418, 2932, 2860, 1750, 1722, 1668, 1159, 1081, 1052 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.86 (3H, t, J=6.8 Hz), 1.01 (3H, s, major), 1.02 (3H, s, minor), 1.15 (3H, s, minor), 1.17 (3H, s, major), 1.20-1.36 (10H, m), 1.47-1.61 (1H, m), 1.70-1.85 (1H, m), 2.10-2.32 (3H, m), 2.55 (1H, br. s, major), 3.09 (1H, br. s, minor), 3.42-3.80 (4H, m), 3.45 (3H, s, minor), 3.46 (3H, s, major), 3.69 (3H, s), 3.98-4.20 (2H, m), 5.20 (1H, s, major), 5.25 (1H, s, minor), 5.96 (1H, s), 9.68 (1H, s, minor) 9.68 (1H, s, major); ¹³C NMR (100 M Hz, CDCl₃) δ 14.0, 16.5, 16.7, 19.0, 19.1, 22.5, 24.3, 28.9, 31.3, 31.3, 31.6, 33.9, 38.8, 38.9, 51.3, 51.3, 51.5, 51.6, 53.9, 66.7, 67.2, 68.2, 68.7, 70.3, 71.0, 71.4, 71.7, 102.2, 102.6, 119.6, 119.7, 1500, 166.3, 171.7, 183.9, 201.8, 202.2.

To diol 204.1 (28.5 mg, 0.0603 mmol) in methylene chloride (2.45 mL) was added pyridine (141.3 μL, 1.75 mmol) followed by TESCl (176 mL, 1.05 mmol) at room temperature. The resulting clear solution was stirred at room temperature for 15 h. Triethylamine (250 μL) was then added and the solution was directly loaded onto a silica column and eluted (5% EtOAc+5% triethylamine/pentane) to afford bis silyl ether 205.1 (42.2 mg, 100%) as an approximately 2.2:1 (β:α) mixture of diastereomers as a colorless oil.

205.1: R_(f) (5% EtOAc+5% triethylamine/pentane)=0.58; IR (film)=2955, 2877, 1754, 1724, 1668, 1462, 1231, 1159, 1107, 1007, 743 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.44-0.65 (12H, m), 0.86 (3H, t, J=6.8 Hz), 0.88-0.98 (18H, m), 0.98 (3H, s, major), 1.00 (3H, s, minor), 1.14 (3H, s, major), 1.15 (3H, s, minor), 1.18-1.32 (10H, m), 1.58-1.71 (1H, m), 1.82-2.00 (1H, m), 2.09-2.21 (3H, m), 3.30-3.71 (3H, m), 3.41 (3H, s, minor), 3.43 (3H, s, major), 3.68 (3H, s, minor), 3.69 (3H, s, major), 3.90-4.10 (2H, m), 5.19 (1H, s, minor), 5.22 (1H, s, major), 5.94 (1H, s, minor), 6.00 (1H, s, major), 9.69 (1H, s, major) 9.70 (1H, s, minor); ¹³C NMR (100 M Hz, CDCl₃) δ 4.3, 4.3, 4.9, 5.2, 6.2, 6.7, 6.8, 6.9, 14.0, 16.4, 16.5, 18.9, 19.0, 22.5, 24.3, 28.9, 29.7, 31.4, 31.6, 31.8, 33.9, 41.2, 41.5, 51.1, 51.2, 51.4, 51.7, 53.9, 67.3, 67.5, 68.0, 69.3, 69.8, 70.3, 71.1, 71.3, 102.1, 102.3, 119.4, 119.5, 150.2, 150.3, 151.0, 166.2, 166.2, 171.7, 202.5, 202.6.

To a solution of diethylmethoxyborane (361 μL, 2.75 mmol) in Et₂O (2.14 mL) was added allylmagnesium bromide (1.0M in Et₂O, 2.50 mmol, 2.50 mL) dropwise at 0° C. The white precipitous mixture was stirred at 0° C. for 60 min. and then allowed to stand for 5 min. A 41.6 μL aliquot (0.5 M allyldiethylborane, 0.021 mmol) of this solution was added dropwise to aldehyde 205.1 (7.3 mg, 0.01 mmol) in 0.5 mL Et₂O at −10° C. After stirring for 30 min., the reaction was quenched with aqueous saturated NH₄Cl (5 mL). The biphasic mixture was then extracted with EtOAc (3×5 mL). The combined organic layers were dried over sodium sulfate, the solution was decanted and then the solvent was removed in vacuo to provide a colorless oil which was taken directly onto the next step.

The crude residue was dissolved in CH₂Cl₂ (1 mL) and treated successively with triethylamine (17.4 μL, 0.125 mmol), 4-dimethylaminopyridine (15.3 mg, 0.125 mmol) and Ac₂O (6 μL, 0.06 mmol) at room temperature. The solution was stirred for 17 h and then pipetted directly onto a short column of silica gel and the products eluted with 7.5% EtOAc/hexanes to afford a diastereomeric mixture of homoallylic acetates (7.9 mg, 97%—2 steps) as a colorless oil.

A portion of the isolated homoallylic acetate (5 mg, 0.0064 mmol) was dissolved in THF (253 μL) and H₂O (25.3 μL) and treated with N-methylmorpholine N-oxide (1.6 mg, 0.014 mmol) followed by OsO₄ (4 wt % in H₂O-16 μL, 0.0025 mmol) at room temperature. The homogeneous solution was stirred for 3 h, and then aqueous saturated sodium bicarbonate (4 mL) and water (1 mL) was added. The biphasic mixture was extracted with EtOAc (5×5 mL). The combined organic layers were dried over sodium sulfate, the solution was decanted and then the solvent was removed in vacuo to provide a colorless oil which was taken directly to the next step.

The resulting residue was immediately dissolved in benzene (0.4 mL) and treated with Et₃N (2.6 μL, 0.025 mmol) at room temperature. Solid Pb(OAc)₄ (4.2 mg, 0.0096 mmol) was quickly added in one portion and the resulting yellow precipitous mixture was stirred vigorously for 30 min. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 12 μL, 0.08 mmol) was introduced and the reaction mixture was stirred for another 30 min. The mixture was added directly to a silica column and eluted (10% EtOAc/Pet. ether) to provide unsaturated aldehyde 206.1 (3.4 mg, 73%—2 steps) as an approximately 2.2:1 (β:α) mixture of diastereomers as a colorless oil.

206.1: R_(f) (10% EtOAc/Pet. ether)=0.42; IR (film)=2955, 2877, 1748, 1723, 1692, 1461, 1229, 1154, 1105, 1008, 743 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.55-0.68 (12H, m), 0.87 (3H, t, J=6.8 Hz), 0.92-1.02 (18H, m), 1.15 (3H, s), 1.18 (3H, s), 1.20-1.34 (10H, m), 1.48-1.60 (2H, m), 1.85-2.03 (1H, m), 2.04-2.23 (2H, m), 2.27-2.39 (1H, m), 3.45-3.68 (2H, m), 3.38 (3H, s, minor), 3.40 (3H, s, major), 3.69 (3H, s, minor), 3.70 (3H, s, major), 3.95-4.05 (1H, m), 4.06-4.17 (1H, m), 5.45 (1H, s, minor), 5.47 (1H, s, major), 5.89 (1H, s), 5.93 (1H, dd, J=16.1/7.8 Hz, major), 5.93 (1H, dd, J=16.1/7.8 Hz, minor), 7.33 (1H, d, J=16.1 Hz, major), 7.36 (1H, d, J=16.1 Hz, minor), 9.53 (1H, d, J=7.8 Hz, major), 9.54 (1H, d, J=7.8 Hz, minor); ¹³C NMR (100 M Hz, CDCl₃) δ 4.3, 4.4, 4.9, 5.4, 6.7, 6.8, 6.9, 7.0, 14.0, 21.7, 21.8, 22.5, 23.6, 23.8, 24.5, 28.9, 28.9, 31.6, 32.5, 32.8, 34.4, 41.2, 41.6, 47.3, 51.2, 51.4, 51.5, 67.3, 67.6, 68.9, 69.2, 69.8, 70.3, 71.0, 71.1, 102.4, 102.6, 117.6, 117.7, 126.7, 151.6, 166.2, 167.2, 171.7, 194.6.

Enal 206.1 (22 mg, 0.03 mmol) was dissolved in acetonitrile (2 mL) and water (205 μL) at room temperature. 48% aqueous HF (388 μL, 12.1 mmol) was added dropwise and the resulting clear solution was stirred at room temperature for 75 min. and was then quenched with a saturated aqueous solution of sodium bicarbonate (15 mL) and water (3 mL). The mixture was extracted with ethyl acetate (5×10 mL). The combined organic layers were dried over sodium sulfate, the solution was decanted and then the solvent was removed in vacuo to provide a crude diol which was taken immediately to the next step.

A 0.75 mM silylating solution was generated by the addition of imidazole (62 mg, 0.91 mmol) and TBSCl (45.6 mg, 0.3 mmol) to methylene chloride (3.9 mL) at room temperature under nitrogen. To the crude diol dissolved in methylene chloride (3.9 mL) and DMF (0.4 mL) was added the above stock solution (1 mL) and stirred at room temperature for 2 h. The solution was then quenched with an aqueous solution of saturated ammonium chloride (10 mL) and extracted with methylene chloride (4×10 mL). The combined organic layers were further washed with brine (10 mL) and then dried over sodium sulfate. The solution was decanted and then the solvent was removed in vacuo. Chromatography (40% EtOAc/pentane) provides the silylated C25 β isomer 207.1b (10.4 mg, 57.4%) along with the silylated C25a isomer 207.1a (4.6 mg, 25.4%) as colorless oils.

207.1b: R_(f) (40% EtOAc/pentane)=0.38; IR (film)=3421, 2930, 2857, 1723, 1691, 1257, 1156, 1110, 837, 779 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.10 (6H, s), 0.87 (3H, t, J=6.8 Hz), 0.92 (9H, s), 1.14 (3H, s), 1.15 (3H, s), 1.18-1.32 (10H, m), 1.49 (1H, t, J=7.2 Hz), 1.66-1.74 (1H, m), 1.84-2.00 (1H, m), 1.98-2.15 (3H, m), 3.49 (1H, dd, J=10.0, 6.0 Hz), 3.66-3.76 (1H, m), 3.70 (3H, s), 3.82-4.00 (2H, m), 4.07 (1H, t, J=6.6 Hz), 4.20 (1H, t, J=10.8 Hz), 5.13 (1H, s), 5.96 (1H, dd, J=16.0/7.7 Hz), 6.03 (1H, s), 7.35 (1H, d, J=16.0 Hz), 9.57 (1H, d, J=7.7 Hz); ¹³C NMR (100 M Hz, CDCl₃) δ −5.3, 14.0, 18.4, 20.0, 22.5, 23.1, 24.4, 25.9, 28.9, 28.9, 31.1, 31.6, 34.5, 39.0, 45.7, 51.3, 67.1, 67.2, 67.9, 72.6, 99.7, 120.7, 127.6, 150.1, 166.1, 171.7, 194.5; [α]^(26.6) _(D)=−27.24° (c=1.04, CH₂Cl₂).

To a solution of acid 408 (Example 2B, 11.2 mg, 0.02 mmol) in toluene (0.9 mL) was added 2,4,6-trichlorobenzoyl chloride (3.2 μl, 0.02 mmol) at room temperature and the mixture was stirred for 45 min. Alcohol 207.1b (10.2 mg, 0.017 mmol) and DMAP (10.4 mg, 0.085 mmol) in toluene (1.4 mL) were added and stirred for 30 min. The mixture was directly loaded onto a silica gel column and eluted (7.5% EtOAc/pentane) to afford the ester 701.1 (15.2 mg, 79%).

701.1: R_(f) (15% ethyl acetate/pentane)=0.29; IR (film)=3492, 2930, 2859, 1725, 1691, 1155, 1112, 837, 777 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.04 (3H, s), 0.05 (3H, s), 0.07 (6H, s), 0.68 (1H, t, J=13.2 Hz), 0.60-0.92 (30H, m), 1.10-1.30 (18H, m), 1.34-1.84 (18H, m), 1.85-2.14 (3H, m), 2.34-2.52 (3H, m), 2.68 (1H, d, J=12.3 Hz), 3.13 (1H, s), 3.34-3.46 (2H, m), 3.60-3.70 (3H, m), 3.68 (3H, s), 3.76-3.86 (2H, m), 3.86-3.96 (1H, m), 4.06 (1H, dt, J=11.9, 2.1 Hz), 4.24-4.34 (1H, m), 5.11 (1H, s), 5.20-5.32 (1H, m), 5.96 (1H, dd, J=16.1, 7.8 Hz), 6.00 (1H, s), 7.42 (1H, d, J=16.1 Hz), 9.58 (1H, d, J=7.8 Hz); ¹³C NMR (100 M Hz, CDCl₃) δ −5.3, −5.3, −4.7, −4.6, 14.0, 18.0, 18.3, 19.2, 20.1, 21.9, 22.3, 22.5, 22.9, 23.6, 23.7, 23.8, 24.3, 24.5, 25.8, 28.9, 28.9, 29.2, 30.9, 31.0, 31.6, 31.8, 31.9, 32.0, 34.5, 35.0, 37.4, 37.4, 43.5, 43.8, 44.7, 45.6, 51.2, 51.3, 59.2, 64.4, 64.9, 65.7, 66.3, 71.1, 72.7, 73.4, 73.8, 99.5, 100.4, 120.5, 127.5, 150.5, 166.4, 166.5, 171.7, 172.1, 194.6; [α]^(24.4) _(D)=−27.42° (c=0.87, CH₂Cl₂).

To seco aldehyde 701.1 (15 mg, 0.013 mmol) in THF (3.7 mL) at room temperature in a plastic flask was added 70% HF/pyridine dropwise. The resulting yellow solution was stirred for 2 hr and was then quenched with a saturated aqueous solution of sodium bicarbonate (12.5 mL) and water (7.5 mL). The biphasic mixture was extracted with ethyl acetate (5×15 mL). The combined organic layers were dried over sodium sulfate, the solution was decanted and then the solvent was removed in vacuo. Chromatography (70% EtOAc/pentane) provides 702.1 (7 mg, 73%) (the corresponding compound of Formula II where X is oxygen) as an amorphous solid.

702.1: R_(f) (80% EtOAc/pentane)=0.29; IR (film)=3454, 3332, 2932, 2858, 1723, 1663, 1138, 976 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.86 (3H, t, J=6.7 Hz), 1.01 (3H, s), 1.17 (3H, s), 1.18-1.38 (12H, m), 1.40-1.66 (7H, m), 1.72-1.88 (3H, m), 1.94-2.14 (3H, m), 2.29 (1H, dt, J=7.5/1.7 Hz), 2.52 (1H, d, J=7.2 Hz), 3.45 (1H, t, J=11.2 Hz), 3.53 (1H, t, J=10.6 Hz), 3.61-3.72 (4H, m), 3.68 (3H, s), 3.88 (3H, t, J=12.4 Hz), 4.02-4.09 (2H, m), 4.10-4.19 (1H, m), 4.48 (1H, d, J=11.6 Hz), 5.02 (1H, d, J=7.6 Hz), 5.10 (1H, s), 5.13 (1H, s), 5.34-5.39 (1H, m), 5.40 (1H, dd, J=15.0/7.6 Hz), 5.97 (1H, d, J=15 Hz), 5.99 (1H, s); ¹³C NMR (100 M Hz, CDCl₃) δ 14.1, 19.3, 22.5, 23.0, 24.4, 24.7, 28.9, 29.0, 31.0, 31.3, 31.4, 31.6, 32.4, 34.6, 35.9, 39.9, 42.5, 42.9, 45.1, 51.1, 64.5, 65.8, 66.3, 68.6, 71.6, 74.1, 75.8, 76.0, 78.7, 98.9, 102.4, 119.9, 125.7, 142.6, 151.7, 167.0, 172.1, 172.6; [α]^(24.0) _(D)=−20.02° (c=0.70, CH₂Cl₂).

3B. Formula IV—Bryostatin Analogue Containing Ether Diester Linker (807)

Enal 801.1, (22 mg, 0.030 mmol) in a tert-butanol-THF solution of 2-methyl 2-butene (1:1, 3 mL) was treated with sodium chlorite (14 mg, 0.152 mmol) and monobasic sodium phosphate (21 mg, 0.152 mmol) in water (0.5 mL). After 1 h, the mixture was diluted with ethyl acetate. The organic layer was dried over sodium sulfate. Column chromatography afforded acid 802.1: R_(f)=(25% ethyl acetate in hexane); [α]²⁵ _(D)=−5.97° (c 0.40, CH₂Cl₂); IR (neat)=2932, 1716, 1644, 1514, 1456, 1374 cm⁻¹; ¹H NMR (500 M Hz, CDCl₃) δ 7.59 (d, J=16.0 Hz, 1H), 7.38 (m, 5H), 7.18 (d, J=8.5 Hz, 2H), 6.85 (d, J=8.5 Hz, 2H), 5.88 (s, 1H), 5.24 (d, J=16.0 Hz, 1H), 5.51 (s, 1H), 5.66 (d, J=11.0 Hz, 1H), 5.60 (d, J=11.0 Hz, 2H), 5.39 (d, J=11.0 Hz, 1H), 4.13 (br, 1H), 3.97 (m, 1H), 3.85 (m, 1H), 3.81 (s, 3H), 3.70 (s, 3H), 3.49 (d, J=14.5 Hz, 1H), 3.26 (s, 3H), 2.41 (t, J=14.5 Hz, 1H), 2.26 (m, 2H), 2.00 (m, 1H), 1.76 (m, 1H), 1.29-1.16 (m, 21H), 0.88 (t, J=7.0 Hz, 3H); ¹³C NMR (125 M Hz, CDCl₃) δ 172.28, 172.05, 166.29, 159.69, 159.10, 152.00, 138.62, 130.51, 129.17, 128.35, 127.61, 127.52, 117.07, 114.72, 113.75, 102.40, 76.19, 74.30, 71.68, 71.11, 70.80, 68.65, 55.23, 51.16, 48.83, 46.87, 36.08, 35.35, 34.13, 32.97, 31.62, 28.89, 24.53, 23.32, 22.50, 22.25, 14.25, 14.07.

Acid 802.1 (22 mg, 0.030 mmol) in toluene (3 mL) was treated with Yamaguchi's reagent (6:L, 0.0395 mmol) and TEA (16:L, 0.122 mmol). After 30 min, alcohol 606 from Example 2F (17 mg, 0.0395 mmol) and DMAP (11 mg, 0.0912 mmol) in toluene (1 mL) was added and stirred for 1 h. The mixture was directly purified by silica gel column to give ester 803.1 (27 mg, 77% yield): R_(f)=(25% ethyl acetate in hexane); [α]²⁵ _(D)=−210.4° (c 0.82, CH₂Cl₂); IR (neat)=2930, 2858, 1720, 1652, 1612, 1514, 1464, 1383, 1250, 1110 cm⁻¹; ¹H NMR (500 M Hz, CDCl₃) δ 7.46 (d, J=16.0 Hz, 1H), 7.36 (m, 5H), 7.18 (d, J=8.5 Hz, 2H), 6.85 (d, J=8.5 Hz, 2H), 5.87 (s, 1H), 5.67 (d, J=16.0 Hz, 1H), 5.50 (s, 1H), 5.29 (q, J=6.0 Hz, 2H), 4.63 (m, 3H), 4.39 (d, J=11.0 Hz, 1H), 4.29 (t, J=11.0 Hz, 1H), 4.20 (m, 2H), 4.12 (t, J=11.0 Hz, 1H), 3.96 (m, 1H), 3.86˜3.66 (m, 7H), 3.47 (m, 5H), 3.25 (s, 3H), 2.53 (d, J=6.5 Hz, 2H), 2.39 (br, 1H), 2.24 (m, 2H), 1.99˜0.85 (m, 26H), 0.09 (s, 3H), 0.07 (s, 3H), 0.04 (s, 9H); ¹³C NMR (125 M Hz, CDCl₃) δ 172.04, 171.07, 167.24, 166.30, 159.10, 157.02, 152.11, 138.68, 130.56, 129.18, 128.35, 128.17, 127.61, 117.04, 115.34, 113.74, 102.42, 88.96, 76.35, 74.48, 71.74, 71.13, 70.90, 68.59, 67.86, 67.34, 67.02, 66.59, 66.61, 61.52, 55.22, 51.12, 46.70, 42.93, 37.24, 36.17, 34.12, 31.64, 29.18, 28.99, 28.88, 25.75, 24.53, 23.45, 22.54, 22.38, 18.00, 17.95, 14.36, 14.06, −1.46, −4.80.

Ester 803.1 (27 mg, 0.0233 mmol) in wet methylene chloride (2 mL) was treated with DDQ (11 mg, 0.0466 mmol) and stirred for 1 h. The mixture was directly purified by silica gel to give the expected alcohol silyl ether product of Formula 804. This silyl ether in acetonitrile-water (10:1, 1.5 mL) was treated with aqueous HF (100 μL, 48%). After 4 h, the mixture was neutralized with aqueous sodium bicarbonate. The aqueous layer was extracted with ethyl acetate. The combined organic layer was dried over sodium sulfate. The crude hydroxy acid product 805.1 was used for the next step.

To a solution of DCC (13 mg, 0.0623 mmol), DMAP HCl (10 mg, 0.0623 mmol) and DMAP (11 mg, 0.089 mmol) was added hydroxy acid 805.1 (7 mg, 0.0089 mmol) in methylene chloride (3 mL) by syringe pump over 10 h. The resultant mixture was loaded directly onto a silica gel column and purified to give C26-O-benzyl ether lactone of Formula 806 where R²⁰ is —O—CO—C₇H₁₅ and R²¹ is ═CH—CO₂Me (806.1) (3.5 mg, 50%). 806.1: R_(f)=(25% ethyl acetate in hexane); [α]²⁵ _(D)=4.74° (c 0.47, CH₂Cl₂); IR (neat)=3746, 3323, 2926, 2851, 1739, 1718, 1624, 1436, 1159 cm⁻¹; ¹H NMR (500 M Hz, CDCl₃) δ 7.39 (br s, 5H), 6.84 (d, J=16.0 Hz, 1H), 6.03 (s, 1H), 5.80 (d, J=16.0 Hz, 1H), 5.41 (br d, J=12.5 Hz, 1H), 5.20 (s, 1H), 5.15 (s, 1H), 4.64 (q, J=12.0 Hz, 2H), 4.47 (q, J=5.5, 11.5 Hz, 1H), 4.31 (m, 3H), 3.99 (t, J=11.0 Hz, 1H), 3.80-3.43 (m, 8H), 2.52 (m, 2H), 2.37˜1.07 (m, 22H), 0.89 (t, J=6.5 Hz, 3H).

To a solution of benzyl ether 806.1 (1 mg) in methylene dichloride (0.5 mL) was added boron trichloride (excess) at −78° C., and the mixture was warmed to −20° C. over 1 h. The reaction mixture was quenched with aqueous sodium bicarbonate. The standard isolation procedure afforded bryostatin analogue 807.1. R_(f)(25% ethyl acetate in hexane); [α]²⁵ _(D)=4.17° (c=0.30, CH₂Cl₂); IR (neat)=3480, 2927, 2856, 2361, 1718, 1281, 1161, 1105 cm⁻¹; ¹H NMR (500 M Hz, PhH) δ 6.82 (d, J=16.5 Hz, 1H), 6.03 (s, 1H), 5.80 (d, J=16.5 Hz, 1H), 5.24 (s, 1H), 5.17 (br s, 2H), 4.36 (m, 3H), 4.01 (t, J=2.5 Hz, 1H), 3.84 (q, J=6.5 Hz, 1H), 3.68 (m, 7H), 3.46 (t, J=9.0 Hz, 1H), 2.57 (m, 2H), 2.33 (m, 2H), 2.18 (m, 1H), 2.11˜1.23 (m, 23H), 0.90 (t, J=10.0 Hz, 3H); ¹³C NMR (125 M Hz, PhH) δ 174.37, 172.11, 171.38, 152.76, 151.21, 121.46, 120.31, 119.94, 99.21, 86.71, 73.84, 73.67, 71.96, 70.03, 68.76, 68.38, 65.14, 51.13, 45.48, 41.06, 35.99, 34.58, 32.91, 31.63, 31.14, 29.00, 28.86, 28.65, 24.68, 23.18, 22.55, 21.13, 19.64, 14.05.

3C. Formula III—Bryostatin Analogue Containing Selected C7 Substituent (705)

To a methyl hemiacetal (30 mg, 0.05 mmol) in acetonitrile (2.8 mL) and water (0.3 mL) was added 48% aq. HF (480 μL) and stirred at room temp. for 2 hrs. The reaction was then quenched with a saturated solution of sodium bicarbonate (5 mL) and extracted with EtOAc (5 mL×4). The combined organics were dried over sodium sulfate and the solvent was removed in vacuo to give expected hemiacetal (a compound according to Formula 303 where R²⁰ is —O—CO—C₇H₁₅ and R²¹ is ═CH—CO₂Me).

To crude acid 513 from Example 2G (47 mg, 0.075 mmol) in toluene (2.6 mL) was added triethylamine (27 μL, 0.2 mmol) and trichlorobenzoyl chloride (12.5 μL, 0.08 mmol), and the mixture was stirred for 90 min. To the resulting solution is added a solution containing the crude hemiacetal and DMAP (30.4 mg, 0.25 mmol) in toluene (4 mL). The resulting precipitous mixture was stirred for 2 h and then added directly to a silica column and eluted (15% EtOAc/pentane to 25% EtOAc/pentane) to afford seco aldehyde 704.1 (41.7 mg, 70%): R_(f) (15% EtOAc/pentane)=0.14; IR (film): 3497.9, 2953.6, 2867.5, 1738.2, 1688.9, 1469.9, 1378.0, 1258.1, 1158.4, 1109.2, 982.5, 835.5, 779.2, 734.9 cm⁻¹; ¹H-NMR (500M Hz, CDCl₃) δ 0.05 (3H, s), 0.08 (3H, s), 0.66 (1H, t, J=13.0 Hz), 0.79-0.88 (21H, m), 0.91-0.96 (1H, m), 0.95 (3H, s), 0.97 (3H, s), 1.15 (3H, s), 1.17-1.29 (15H, m), 1.36-1.50 (6H, m), 1.59-1.78 (5H, m), 1.87-2.13 (5H, m), 2.38 (1H, dquin, J=6.8, 1.3 Hz), 2.51-2.64 (5H, m), 3.31 (1H, s), 3.60-3.68 (2H, m), 3.68 (3H, s), 3.76-3.80 (1H, m), 3.78 (1H, d, J=11.0 Hz), 3.83-3.93 (1H, m), 3.99-4.07 (1H, m), 4.00 (1H, d, J=11.0 Hz), 4.07 (2H, d, J=3.0 Hz), 4.43-4.48 (1H, m), 4.56 (1H, d, J=12.0 Hz), 4.63 (1H, d, J=12.0 Hz), 4.96 (1H, dd, J=9.5, 2.0 Hz), 5.12 (1H, s), 5.41-5.44 (1H, m), 5.95 (1H, dd, J=16.0, 8.0 Hz), 6.00 (1H, d, J=2.0 Hz), 7.27-7.35 (5H, m), 7.38 (1H, d, J=16.0 Hz), 9.49 (1H, d, J=8.0 Hz); ¹³C-NMR (125M Hz, CDCl₃) δ −5.1, −4.8, 14.0, 15.0, 17.9, 19.0, 19.8, 20.2, 21.6, 21.7, 22.4, 22.5, 22.8, 23.7, 24.3, 24.4, 25.7, 28.8, 28.8, 28.9, 30.9, 31.1, 31.6, 34.5, 34.9, 35.4, 37.2, 37.5, 38.1, 40.7, 41.6, 42.0, 45.7, 51.1, 51.2, 58.9, 65.6, 65.7, 66.1, 70.8, 71.1, 71.4, 72.5, 73.1, 74.8, 99.5, 100.5, 120.7, 127.5, 127.6, 127.8, 128.4, 138.2, 150.3, 166.2, 166.3, 167.3, 170.5, 171.7, 171.8, 194.6; HRMS calc'd for C₆₅H₁₀₃ClO₁₇Si (+1Na): 1241.6532. found: 1241.6551; [α]^(27.0) _(d): −31.67° (c 4.17, CH₂Cl₂).

To 704.1 (38 mg, 0.031 mmol) in THF (9.5 mL) was added freshly dried 4 Angstrom molecular sieve beads (57 beads) and 70% HF/pyridine (2.3 mL), and the resulting solution was stirred for 45 min. in a plastic flask. The reaction was then poured into a saturated solution of sodium bicarbonate (95 mL) and diluted with EtOAc (30 mL). The layers were separated and the aqueous layer was extracted with EtOAc (40 mL×3). The combined organic layers were dried over sodium sulfate and the solvent was removed in vacuo. Silica chromatography (40% to 100% EtOAc/pentane) afforded 705.1 (13.4 mg, 45%) and a putative diol 705.1a without the 3-hydroxy TBS protecting group (9.0 mg, 30%).

To putative diol 705.1a (4.8 mg, 4.95 μmol) in THF (0.5 mL) was added freshly dried 4 A molecular sieve beads (3 beads) and 70% HF/pyridine (0.1 mL) and the resulting solution was stirred for 40 min. in a plastic flask. The reaction was then poured into a saturated solution of sodium bicarbonate (5 mL) and diluted with EtOAc (30 mL). The layers were separated and the aqueous layer was extracted with EtOAc (4 mL×4). The combined organics were dried over sodium sulfate and the solvent was removed in vacuo. Chromatography (30% to 100% EtOAc/pentane) afforded 705.1 (a compound of Formula III where R³ is OH, R⁵ is ═O, R⁸ is t-Bu-chloroacetate, R⁹ is H, R²⁰ is —O—CO—C₇H₁₅, R²¹ is ═CH—CO₂Me and R²⁶ is Me). (1.1 mg, effectively adding 7% to above yield=52%). 705.1: R_(f) (30% EtOAc/pentane)=0.23; IR (film): 3391.9, 2929.4, 2856.8, 1731.9, 1664.5, 1434.2, 1375.1, 1249.6, 1160.5, 1133.2, 1098.3, 982.1, 735.8 cm⁻¹; ¹H-NMR (500M Hz, CDCl₃) δ 0.87 (3H, t, J=7.0 Hz), 0.93 (3H, s), 0.96 (3H, s), 1.03 (3H, s), 1.18 (3H, d, J=6.0 Hz), 1.20 (3H, s), 1.22-1.30 (10H, m), 1.51 (1H, br. d, J=12.5 Hz), 1.69-1.81 (3H, m), 2.00-2.08 (4H, m), 2.29-2.34 (3H, m), 2.41 (1H, dd, J=12.5, 4.0 Hz), 2.63 (1H, dd, J=14.3, 2.5 Hz), 2.82 (1H, dd, J=12.5, 4.5 Hz), 3.58 (1H, d, J=11.0 Hz), 3.61-3.64 (1H, m), 3.67-3.74 (2H, m), 3.68 (3H, s), 3.80 (1H, d, J=11.0 Hz), 3.82 (1H, t, J=12.0 Hz), 3.95-3.99 (1H, m), 3.97 (1H, d, J=11.0 Hz), 4.05-4.10 (1H, m), 4.08 (2H, s), 4.34 (1H, s), 4.39-4.44 (1H, m), 4.53 (1H, d, J=12.0 Hz), 4.65 (1H, d, J=12.0 Hz), 5.12 (1H, s), 5.19 (1H, dd, J=12.0, 3.0 Hz), 5.41 (1H, dd, J=16.0, 7.3 Hz), 5.50 (1H, ddd, J=12.3, 4.0, 3.0 Hz), 5.98 (1H, d, J=2.0 Hz), 6.08 (1H, d, J=16.0 Hz), 7.29-7.38 (5H, m); ¹³C-NMR (125M Hz, CDCl₃) δ 14.0, 15.3, 19.4, 20.0, 21.4, 22.5, 23.9, 24.7, 28.8, 29.0, 31.2, 31.6, 32.6, 34.5, 34.6, 37.1, 38.5, 40.8, 42.2, 43.2, 45.0, 51.1, 65.0, 65.8, 66.5, 71.0, 71.1, 71.1, 73.2, 74.0, 75.0, 75.5, 98.7, 101.2, 119.7, 126.9, 127.7, 127.8, 128.4, 138.2, 142.7, 151.4, 166.8, 167.2, 170.1, 170.1, 172.1; HRMS calc'd for C₄₉H₇₁ClO₁₆ (+1Na): 973.4350. found: 973.4328; [α]^(25.0) _(d): −3.04° (c 1.34, CH₂Cl₂).

To 705.1 (7.4 mg, 7.785 μmol) in THF (0.66 mL) was added thiourea (66 mg, 0.84 mmol) and the resulting slurry was stirred at room temperature for 3 days. The reaction was then added directly to a silica column and eluted (50% to 58% to 70% EtOAc/pentane) to afford 705.2 (a compound of Formula III where R³ is OH, R⁵ is ═O, R⁸ is t-Bu-OH, R⁹ is H, R²⁰ is —O—CO—C₇H₁₅, R²¹ is ═CH—CO₂Me and R²⁶ is Me). (6.3 mg, 92%). 705.2: R_(f) (50% EtOAc/pentane)=0.18; IR (film): 3423.1, 2926.7, 2856.8, 1726.4, 1659.2, 1376.1, 1253.2, 1159.3, 1098.3, 980.8, 799.1, 729.7 cm⁻¹; ¹H-NMR (300M Hz, CDCl₃) δ 0.77 (3H, s), 0.86 (3H, t, J=10.8 Hz), 0.96 (3H, s), 1.03 (3H, s), 1.18 (3H, d, J=8.1 Hz), 1.20 (3H, s), 1.22-1.34 (10H, m), 1.68-1.82 (3H, m), 2.01-2.12 (3H, m), 2.18-2.33 (3H, m), 2.45 (1H, dd, J=12.6, 4.5 Hz), 2.62-2.67 (2H, m), 2.85 (1H, dd, J=12.6, 3.6 Hz), 3.09-3.21 (2H, m), 3.60-3.88 (5H, m), 3.68 (3H, s), 3.93-4.00 (1H, m), 4.09 (1H, dd, J=11.1, 4.5 Hz), 4.35 (1H, s), 4.35-4.60 (1H, m), 4.52 (1H, d, J=12.0 Hz), 4.65 (1H, d, J=12.0 Hz), 5.06 (1H, d, J=9.6 Hz), 5.12 (1H, s), 5.18 (1H, d, J=7.2 Hz), 5.42 (1H, dd, J=15.9, 7.2 Hz), 5.44-5.51 (1H, m), 5.98 (1H, s), 6.08 (1H, d, J=15.9 Hz), 7.35-7.39 (5H, m); ¹³C-NMR (125M Hz, CDCl₃) δ 14.1, 15.3, 18.4, 19.4, 22.4, 22.5, 23.9, 24.7, 28.9, 29.0, 29.7, 31.2, 31.6, 32.7, 34.5, 34.7, 36.8, 39.7, 42.3, 43.2, 45.0, 51.1, 65.0, 65.9, 66.5, 69.0, 71.1, 71.2, 74.0, 75.0, 77.2, 98.7, 101.3, 119.7, 126.9, 127.7, 127.9, 128.4, 138.2, 142.8, 151.5, 166.9, 170.1, 171.8, 172.1; HRMS calc'd for C₄₇H₆₉O₁₅ (+1Na): 897.4595. found: 897.4612; [α]^(25.0) _(d): −90.50° (c 0.47, CH₂Cl₂).

To myristic acid (10 mg, 0.044 mmol) in toluene (2.14 mL) at rt under nitrogen is added triethylamine (23.3 μL, 0.175 mmol) followed by 2,4,6-trichlorobenzoylchloride (6.8 μL, 0.044 mmol). The resulting solution was stirred for 45 min. An aliquot of this solution (63 μL, 0.0013 mmol) was added to a solution of 705.2 (1 mg, 0.00114 mmol) and DMAP (0.7 mg, 0.0059 mmol) in toluene (500 μL). The slightly yellow, cloudy solution is stirred at rt for 30 min. and then added directly to a silica column and eluted (30% EtOAC/pentane). The eluted material is then re-chromatographed (30% EtOAC/pentane). The resulting material was then dissolved in EtOAc (500 μL) and Pearlman's catalyst (2 mg) was added. The resulting suspension was evacuated and re-filled with hydrogen (5 times) while the reaction was stirred vigorously. After 30 min., the reaction is added directly to a silica column and eluted (EtOAc). This provided C7 myristate analogue 705.3 (360 μg, 32%—2 steps) (a compound of Formula III where R³ is OH, R⁵ is ═O, R⁸ is t-Bu-myristate, R⁹ is H, R²⁰ is —O—CO—C₇H₁₅, R²¹ is ═CH—CO₂Me and R²⁶ is Me). R_(f) (40% EtOAc/pentane)=0.19. IR (film) 3256.8, 2915.8, 2845.2, 1746.4, 1722.9, 1158.4, 1029.0, 864.4, 793.8 cm⁻¹; ¹H-NMR (500M Hz, CDCl₃) δ 0.86-0.94 (12H, m), 1.05 (3H, s), 1.14-1.38 (38H, m), 1.59-1.63 (2H, m), 1.63-1.88 (3H, m), 2.03-2.07 (3H, m), 2.31-2.42 (4H, m), 2.47 (1H, dd, J=12.8, 3.8 Hz), 2.72-2.74 (2H, m), 2.86 (1H, dd, J=12.5, 5.0 Hz), 3.70 (3H, s), 3.62-3.75 (3H, m), 3.78-3.87 (3H, m), 3.99-4.04 (1H, m), 4.09-4.14 (1H, m), 4.37-4.50 (2H, m), 5.15 (1H, s), 5.16 (1H, d, J=7.4 Hz), 5.21 (1H, d, J=12.0 Hz), 5.33-5.34 (1H, m), 5.43 (1H, dd, J=15.6, 7.4 Hz), 6.01 (1H, s), 6.07 (1H, d, J=15.6 Hz). ¹³C-NMR (500M Hz, CDCl₃) δ 14.1, 19.9, 20.5, 21.1, 22.7, 23.2, 23.9, 24.7, 24.7, 24.9, 26.7, 29.1, 29.3, 29.4, 29.4, 29.7, 30.2, 31.1, 31.6, 31.9, 33.5, 33.7, 34.3, 34.5, 35.8, 37.2, 38.4, 42.3, 43.3, 45.0, 51.1, 65.0, 65.9, 66.6, 69.4, 70.0, 73.3, 73.5, 74.0, 75.9, 77.2, 98.7, 101.2, 119.8, 126.9, 128.8, 142.7, 151.3, 166.8, 170.4, 170.9, 172.4. HRMS (FAB) calc'd for C₅₄H₉₀O₁₆Na: 1017.6133. found: 1017.6127. [α]^(21.1) _(D)=−7.14° (c 0.035, CH₂Cl₂).

3D. Formula V—Bryostatin Analogue Containing Diester Linker (903.1)

To a solution of silyl ether 111 from Example IC (1.17 g, 1.44 mmol) and pyridine (2.07 mL, 25.65 mmol) in 12 mL THF in a polypropylene vial was added HF/pyridine complex (0.83 mL, 28.83 mmol) at rt. The solution was stirred for 24 hours and diluted with EtOAc. The organic layer was washed with sat. CuSO₄ and brine, dried over Na₂SO₄ and concentrated in vacuo to afford the corresponding alcohol (not shown) as a pale yellow oil. To the alcohol (24.6 mg, 0.036 mmol) in methylene chloride (0.7 mL) was added DMAP (24.5 mg, 0.201 mmol) followed by succinic anhydride (8.6 mg, 0.086 mmol) at rt. The solution was heated to 42° C. for 3 hours and then slowly cooled to rt. Col. chromatography (40% EtOAc+1% AcOH/hexane) provided crude 901.1 (28.6 mg, 0.0353 mmol).

To crude 901.1 (28.6 mg) in methylene chloride (0.8 mL) and water (9.5 μL) was added DDQ (10.4 mg, 0.046 mmol) at rt. After stirring for 2 hours, the reaction was quenched with a saturated aqueous solution of ammonium chloride and extracted with EtOAc (3×5 mL). The combined organics were then dried over sodium sulfate and the solvent was removed in vacuo. Chromatography (40% EtOAc+1% AcOH/hexane) provided seco acid 902.1 (21.9 mg, 0.032 mmol, 91% in 2 steps).

To 902.1 (5 mg, 7.38 μmol) in acetonitrile (0.4 mL) and water (42 μL) at rt was added 48% aqueous HF dropwise (24 μL, 0.738 mmol). The reaction was stirred for 40 min. and then quenched with a saturated aqueous solution of sodium bicarbonate (5 mL). The layers were separated and then the aqueous layer was extracted with EtOAc (6×5 mL). The combined organics were dried over sodium sulfate and then the solvent was removed in vacuo. The resulting clear oil (not shown) was then used immediately in the next step.

To DMAP (9 mg, 0.074 mmol) and DMAP.HCl (8.2 mg, 0.052 mmol) in methylene chloride (1.4 mL) was added DCC (10.6 mg, 0.052 mmol) at rt. The clear oil from the preceding step in methylene chloride (2.2 mL) was then added over 3 h. The resulting mixture was stirred at rt for 4 h and then quenched with a saturated aqueous solution of sodium bicarbonate (5 mL). The layers were separated and then the aqueous layer was extracted with EtOAc (3×5 mL). The combined organics were then dried over sodium sulfate and the solvent was removed in vacuo. Silica gel chromatography (30% EtOAc/hexane) provided corresponding crude macrocycle (not shown) as a colorless oil.

The crude macrocycle from the preceding step was dissolved in ethyl acetate (2.6 mL) and Pd(OH)₂/C (2.4 mg, 20% wt. on carbon) was added. The resulting suspension was evacuated and refilled with 1 atm. hydrogen gas (×5) and was vigorously stirred under a hydrogen atmosphere for 3 hours. The crude mixture was pipetted directly onto a silica gel column and the product was eluted (50% EtOAc/hexane) to afford of bryostatin analogue 903.1 (0.3 mg, 7%—3 steps) (a compound of Formula V where p is 2, R²⁰ is —O—CO—C₇H₁₅, R²¹ is ═CH—CO₂Me and R²⁶ is Me) as a white solid.

Example 4 Bryostatin Analogues Containing Selected C20 Ester Substituent

This example illustrates methods for preparing bryostatin compounds and analogues that contain selected ester substituents at C20.

4A. Acetyl C20 Ester (702.2)

To a solution of enal 13 (180 mg, 0.03 mmol) in 0.5 mL of MeOH at rt was added pyridinium p-toluenesulfonate (PPTS, 2 mg, catalytic) and trimethylorthoformate (5 drops). The progress of the reaction was monitored by thin layer chromatography (TLC). After 30 min the reaction was quenched with 1.0 mL Et₃N. The solvent was removed under reduced pressure to afford the expected crude dimethylacetal product (not shown). This product was immediately dissolved in MeOH (0.5 mL) and K₂CO₃ (3 mg, catalytic). The progress of the reaction was monitored by TLC. After 30 min the reaction was quenched. The solution was quenched with sat. NaHCO₃, diluted with EtOAc (10 mL), washed with H₂O, dried over MgSO₄ and concentrated under reduced pressure to afford the crude C20 free hydroxyl product (not shown). The crude product was immediately dissolved in methylene chloride (0.5 mL) and acetic anhydride (0.3 mL, excess) and DMAP (5 mg, catalytic) was added at rt. The progress of the reaction was monitored by TLC. After 30 min the reaction was quenched. The solution was quenched with sat. NaHCO₃, diluted with EtOAc (10 mL), washed with H₂O, dried over MgSO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel with 20% EtOAc-hexanes as eluant affording 14 mg (82% for four steps) of dimethylacetal 37. R_(f) (20% ethyl acetate/hexanes)=0.17; R_(f) (25% EtOAc/hexanes)=0.44; IR 2927, 2859, 1744, 1719, 1687, 1514, 1249, 1156, 1103, 1079, 1037 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.85 (6H, m) 1.16-1.30 (10H, m), 1.14 (3H, s), 1.18 (3H, s), 1.75 (1H, m), 1.98-2.17 (3H, m), 2.32 (1H, m), 3.27 (3H, s), 3.52 (1H, d, J=16.5 Hz), 3.68 (3H, s), 3.79 (3H, s), 3.87 (1H, m), 3.95 (1H, m), 4.09 (1H, m), 4.47 (2H, ABq, J=11.4 Hz), 5.41 (1H, s), 5.88 (1H, s), 5.91 (1H, dd, J=16.2, 7.7 Hz), 6.83 (2H, d, J=8.7 Hz), 7.16 (2H, d, J=8.7 Hz), 7.27-7.35 (6H, m), 9.44 (1H, d, J=7.7 Hz, C15); ¹³C NMR (75 M Hz, CDCl₃) δ 14.1, 21.7, 22.6, 24.0, 24.6, 28.9, 29.0, 31.7, 32.6, 34.5, 36.2, 47.5, 51.3, 51.5, 55.4, 69.2, 71.3, 71.8, 74.4, 76.3, 102.7, 114.1, 118.2, 127.1, 127.7, 127.9, 128.7, 129.5, 130.7, 138.9, 151.6, 159.6, 166.6, 167.3, 172.1, 195.0; [α]_(D) ²⁰=−0.7° (c 1.7, CH₂Cl₂).

To a solution of dimethylacetal 37 (14 mg, 0.02 mmol) in 0.6 mL 1% aqueous CH₂Cl₂ was added solid 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 10 mg, 0.03 mmol) at rt. The mixture was stirred for 2 h, pipetted directly onto a column of silica gel, and the product eluted with 35% EtOAc/hexanes to provide intermediate alcohol 303.1 (10 mg, 91%) as a colorless oil: R_(f) (35% EtOAc/hexanes)=0.22; IR 3528, 2930, 2858, 1745, 1720, 1686, 1458, 1437, 1380 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.86 (3H, t, J=6.9 Hz), 1.13 (3H, s), 1.17 (3H, s), 1.25 (10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12 (2H, m), 2.35 (1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H, s, C19 OCH₃), 3.45 (1H, m), 3.68 (3H, s), 3.82 (1H, s), 4.24 (1H, m), 4.55 (2H, ABq, J=11.4 Hz), 5.47 (1H, s), 5.86 (1H, s), 5.91 (1H, dd, J=15.9, 7.5 Hz), 7.29 (1H, d, J=15.9 Hz), 7.34 (5H, s), 9.52 (1H, d, J=7.5 Hz); ¹³C-NMR (75 M Hz, CDCl₃) δ 13.6, 15.3, 21.6, 22.4, 23.5, 24.7, 28.6, 28.8, 31.5, 32.8, 34.1, 39.5, 47.4, 51.1, 51.2, 68.3, 70.9, 71.0, 71.1, 78.8, 102.4, 117.4, 126.8, 127.9, 128.0, 128.5, 138.1, 151.8, 166.4, 167.3, 171.4, 194.5; [α]_(D) ²⁰=−21.00 (c 1.0, CH₂Cl₂).

Alcohol 303.1 (10 mg, 0.02 mmol) was dissolved in 1.1 mL CH₃CN/H₂O (9:1) and treated with 48% aqueous HF (200 μL, 300 mol % excess) at rt. The resulting mixture was stirred for 1 h, quenched with sat. NaHCO₃ and diluted with 10 mL EtOAc. The aqueous layer was separated and extracted with EtOAc (2×). The combined organics were dried over Na₂SO₄ and concentrated in vacuo to afford crude hemiketal enal 304.1 as a colorless oil. The crude product was purified by column chromatography on silica gel with 35% EtOAc-hexanes as eluant affording 8 mg (89%) of hemiketal enal 304.1. R_(f) (35% EtOAc/hexanes)=0.15; IR 3528, 2930, 2858, 1745, 1720, 1686, 1458, 1437, 1380 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.86 (3H, t, J=6.9 Hz, octanoate Me), 1.13 (3H, s, C18 Me), 1.17 (3H, s, C18 Me), 1.25 (10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12 (2H, m), 2.35 (1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H, s, C19 OCH₃), 3.45 (1H, m), 3.68 (3H, s, methyl ester), 3.82 (1H, s), 4.24 (1H, m), 4.44 (1H, d, J=11.1 Hz, CH₂Ph), 4.69 (1H, d, J=11.4 Hz, CH₂Ph), 5.47 (1H, s, C20), 5.86 (1H, s, C34), 5.91 (1H, dd, J=15.9, 7.5 Hz, C16), 7.29 (1H, d, J=15.9 Hz, C17), 7.34 (5H, s, Ph), 9.52 (1H, d, J=7.5 Hz, C15); ¹³C-NMR (75 M Hz, CDCl₃) δ 13.8, 15.4, 21.7, 22.3, 23.6, 24.3, 28.7, 28.8, 31.4, 32.7, 34.2, 39.5, 47.3, 51.1, 51.2, 68.3, 70.8, 71.0, 71.1, 78.1, 102.3, 117.4, 126.8, 128.0, 128.1, 128.6, 138.1, 151.8, 166.4, 167.1, 171.7, 194.7; [α]_(D) ²⁰=19.0° (c 1.4, CH₂Cl₂).

Carboxylic acid 407 (Example 2B, 15 mg, 0.03 mmol) and Et₃N (16.5 μL, 0.12 mmol) were dissolved in 300 μL toluene and treated with 2,4,6-trichlorobenzoylchloride (4.8 μL, 0.03 mmol) dropwise at rt. After 1 h at rt, a toluene solution of freshly prepared 304.1 and 4-dimethylaminopyridine (14 mg, 0.12 mmol) was added gradually and stirring was continued for 40 min. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 20% EtOAc/hexanes to provide ester 701.1 as a colorless oil (15 mg, 63%). R_(f) (35% EtOAc/hexanes)=0.71; IR 3487, 2927, 2856, 1723, 1689, 1455, 1379, 1228, 1156, 1113, 1084, 1032, 981 cm⁻¹; ¹H NMR (300 M Hz, C₆D₆) δ 0.80 (1H, t, J=12.7 Hz), 0.93 (3H, t, J=7.0 Hz), 0.97 (3H, d, J=6.6 Hz), 1.10-1.36 (24H, m), 1.36-1.85 (21H, m), 1.91-2.13 (6H, m), 2.39 (1H, t, J=12.8 Hz), 2.79 (1H, d, J=13.2 Hz), 2.93 (1H, m), 3.05 (1H, m), 3.29 (3H, s), 3.33 (1H, s), 3.37-3.48 (2H, m), 3.80 (1H, dd, J=11.4, 5.1 Hz), 3.95-4.05 (2H, m), 4.15 (1H, td, J=10.8, 0.9 Hz), 4.23 (1H, d, J=13.5 Hz), 4.40 (1H, d, J=12.0 Hz), 4.47 (1H, d, J=12.0 Hz), 5.57 (1H, s), 5.64 (1H, dd, J=10.6, 4.6 Hz), 6.05 (1H, dd, J=16.1, 7.6 Hz), 6.39 (1H, s), 7.10-7.35 (5H, m), 7.45 (1H, d, J=16.1 Hz), 9.60 (1H, d, J=7.6 Hz); ¹³C-NMR (75 M Hz, C₆D₆) δ 14.1, 15.1, 19.3, 20.1, 21.3, 22.3, 22.4, 22.7, 23.8, 24.0, 24.6, 24.6, 29.0, 29.0, 29.3, 31.3, 31.6, 31.8, 34.2, 34.5, 35.2, 35.7, 36.2, 37.6, 43.5, 45.7, 51.6, 59.1, 64.9, 66.7, 71.1, 72.0, 72.9, 74.1, 75.3, 77.1, 100.2, 100.6, 121.2, 127.2, 127.3, 127.9, 128.6, 138.9, 151.2, 164.5, 166.3, 171.5, 175.1, 193.4; [α]²⁰ _(D)=−19° (c 1.5, CH₂Cl₂).

To ester 701.1 (13 mg, 0.02 mmol) in THF (0.5 mL) was added pyridine (360 μL, 0.45 mmol) followed by 70% HF/pyridine (144 μL, 500 mol % excess) and stirred for 20 hours. The reaction was then quenched with a saturated solution of sodium bicarbonate. The biphasic mixture was extracted with ethyl acetate (×4) and the combined organics were dried over sodium sulfate. The solvent was removed in vacuo to provide crude C3 hydroxyester 701.2. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 35% EtOAc/hexanes to provide ester 701.2 as a colorless oil (9 mg, 82%). R_(f) (40% EtOAc/hexanes)=0.19; IR 3522, 2927, 2857, 1724, 1664, 1230, 1158, 1136, 1107, 979 cm⁻¹; ¹H NMR (400 M Hz, C₆D₆) δ 0.84 (3H, t, J=5.4 Hz), 0.88-0.96 (5H, m), 1.00 (3H, d, J=4.8 Hz), 1.02-1.55 (27H, m), 1.63-1.81 (2H, m), 1.82-1.94 (2H, m), 2.03 (1H, brt, J=5.2 Hz), 2.19-2.27 (1H, m), 2.34 (1H, dt, J=9, 1.5 Hz), 2.94-3.01 (2H, m), 3.22 (1H, s), 3.58 (1H, br d, J=3.6 Hz), 3.68-3.74 (1H, m), 3.84-3.88 (1H, m), 3.94 (1H, dd, J=8.6, 3.1 Hz), 4.23 (1H, dd, J=10.4, 1.7 Hz), 4.31 (1H, brt, J=8.1 Hz), 5.36-5.41 (1H, m), 5.50 (1H, s), 5.61 (1H, d, J=5.4 Hz), 6.00 (1H, dd, J=12.0, 5.4 Hz), 6.36 (1H, s), 6.53 (1H, d, J=12.0 Hz); ¹³C NMR (100 M Hz, C₆D₆) δ 14.2, 19.4, 19.8, 22.4, 22.9, 24.0, 24.5, 25.0, 29.1, 29.2, 30.2, 31.8, 31.9, 32.0, 33.1, 34.6, 34.9, 35.4, 36.5, 43.6, 45.6, 50.7, 66.1, 66.7, 69.6, 73.2, 74.7, 75.8, 76.3, 77.5, 98.7, 102.6, 120.5, 140.1, 151.2, 151.5, 166.5, 171.5, 174.2 [α]²⁰ _(D)=−13.5° (c 0.9, CDCl₃).

To a solution of C3 hydroxyester 701.2 (8 mg, 0.01 mmol) in 2.0 mL CH₂Cl₂ was added 4 Å molecular sieves and the mixture was aged for 20 min. 45-50 beads of Amberlyst-15 sulfonic acid resin were added and the mixture was stirred at rt for 2 h. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 35% EtOAc/hexanes to provide the expected macrocyclic product (not shown) as a colorless oil (5 mg, 83%). R_(f) (35% EtOAc/hexanes)=0.21; IR 3522, 2927, 2857, 1724, 1664, 1230, 1158, 1136, 1107, 979 cm⁻¹; ¹H NMR (400 M Hz, C₆D₆) δ 0.84 (3H, t, J=5.4 Hz), 0.88-0.96 (5H, m), 1.00 (3H, d, J=4.8 Hz), 1.02-1.55 (27H, m), 1.63-1.81 (2H, m), 1.82-1.94 (2H, m), 2.03 (1H, brt, J=5.2 Hz), 2.19-2.27 (1H, m), 2.34 (1H, dt, J=9, 1.5 Hz), 2.94-3.01 (2H, m), 3.22 (1H, s), 3.58 (1H, br d, J=3.6 Hz), 3.68-3.74 (1H, m), 3.84-3.88 (1H, m), 3.94 (1H, dd, J=8.6, 3.1 Hz), 4.23 (1H, dd, J=10.4, 1.7 Hz), 4.31 (1H, brt, J=8.1 Hz), 5.36-5.41 (1H, m), 5.50 (1H, s), 5.61 (1H, d, J=5.4 Hz), 6.00 (1H, dd, J=12.0, 5.4 Hz), 6.36 (1H, s), 6.53 (1H, d, J=12.0 Hz); ¹³C NMR (100 M Hz, C₆D₆) δ 14.2, 19.4, 19.8, 22.4, 22.9, 24.0, 24.5, 25.0, 29.1, 29.2, 30.2, 31.8, 31.9, 32.0, 33.1, 34.6, 34.9, 35.4, 36.5, 43.6, 45.6, 50.7, 66.1, 66.7, 69.6, 73.2, 74.7, 75.8, 76.3, 77.5, 98.7, 102.6, 120.5, 140.1, 151.2, 151.5, 166.5, 171.5, 174.2.

The macrocyclic product from the preceding step was dissolved in 0.5 mL EtOAc and 2.2 mg Pd(OH)2 (20% wt. on carbon) was added. The resulting suspension was vigorously stirred under balloon pressure of hydrogen gas for 35 min. The crude mixture was pipetted directly onto a column of silica gel and the product was eluted with 60% EtOAc/hexanes to afford acetate analogue 702.2 (4 mg, 93%) (a compound of Formula II where R³ is OH, R²⁰ is —O—CO-Me, R²¹ is ═CH—CO₂Me, R²⁶ is Me and X is oxygen) as a white semi-solid. R_(f) (50% EtOAc/hexanes)=0.16; IR 3522, 2927, 2857, 1724, 1664, 1230 IR (neat)=3455, 3319, 2929, 2856, 1735, 1380, 1230, 1138, 976 cm⁻¹; ¹H NMR (500 M Hz, CDCl₃) δ 0.37 (3H, br. s), 0.77-0.92 (14H, m), 1.06 (3H, d, J=6.4 Hz), 1.07 (1H, t, J=11.0 Hz), 1.10-1.28 (5H, m), 1.27 (3H, s), 1.50 (3H, s), 1.57-1.78 (4H, m), 2.03 (2H, t, J=7.4 Hz), 2.17 (1H, dd, J=9.9, 0.5 Hz), 2.37 (1H, m), 2.40 (1H, m), 2.85 (1H, t, J=11.2 Hz), 2.96 (1H, t, J=10.8 Hz), 3.15 (3H, s), 3.68-3.75 (3H, m), 3.91 (1H, dd, J=11.2, 4.0 Hz), 4.12 (1H, t, J=9.7 Hz), 4.35 (1H, dd, J=13.9, 2.3 Hz), 4.48 (1H, td, J=11.0, 2.8 Hz), 4.70 (1H, d, J=12.1 Hz), 5.44 (1H, quint., J=4.8 Hz), 5.54 (1H, d, J=7.3 Hz), 5.69 (1H, s), 5.76 (1H, s), 5.85 (1H, dd, J=16.1, 7.5 Hz), 6.40 (1H, d, J=1.8 Hz), 6.50 (1H, d, J=15.9 Hz); ¹³C NMR (125 M Hz, CDCl₃) δ 14.3, 19.6, 12.0, 22.9, 23.2, 24.9, 25.0, 29.2, 29.2, 31.4, 31.5, 31.9, 32.9, 34.7, 36.3, 39.9, 42.6, 43.2, 45.4, 50.5, 65.2, 66.2, 67.0, 70.4, 74.2, 74.7, 75.3, 75.9, 78.5, 99.7, 103.1, 120.5, 142.5, 152.5, 171.6, 172.5; [α]²⁵ _(D)=−9.0° (c=0.36, CDCl₃).

4B. Heptanoate C20 Ester (702.3)

To solution of the enal of Formula 305 (in which R²⁰ is C₇H₁₅), (224 mg, 0.04 mmol) in 0.5 mL of MeOH at rt was added PPTS (2 mg, catalytic) and trimethylorthoformate (1 drop). The progress of the reaction was monitored by TLC. After 30 min the reaction was quenched with 1.0 mL Et₃N. The solvent was removed under reduced pressure to afford the corresponding crude dimethylacetal product of Formula 306. This product was immediately dissolved in MeOH (2.0 mL) and K₂CO₃ (3 mg, catalytic). The progress of the reaction was monitored by TLC. After 30 min the reaction was quenched. The solution was quenched with sat. NaHCO₃, diluted with EtOAc (10 mL), washed with H₂O, dried over MgSO₄ and concentrated under reduced pressure to afford the corresponding crude C20 free hydroxyl product of Formula 307.

Heptenoic acid (6 mg, 0.05 mmol) and Et₃N (21 μL, 0.16 mmol) were dissolved in 600 μL toluene and treated with 2,4,6-trichlorobenzoylchloride (7.0 μL, 0.05 mmol) dropwise at rt. After 1 h at rt, a toluene solution of the freshly prepared C20 free hydroxyl product of Formula 307 and 4-dimethylaminopyridine (DMAP, 20 mg, 0.17 mmol) was added gradually and stirring was continued for 40 min. After 30 min the reaction was quenched. The solution was quenched with sat. NaHCO₃, diluted with EtOAc (10 mL), washed with H₂O, dried over MgSO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel with 20% EtOAc-hexanes as eluant affording 21 mg (84%) of the corresponding dimethylacetal, C20 heptenoate product of Formula 308.1. R_(f) (20% ethyl acetate/hexanes)=0.22; IR 2927, 2859, 1744, 1719, 1687, 1514, 1249, cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.85 (6H, m) 1.16-1.30 (10H, m), 1.14 (3H, s), 1.18 (3H, s), 1.75 (1H, m), 1.98-2.17 (3H, m), 2.32 (1H, m), 3.27 (3H, s), 3.52 (1H, d, J=16.5 Hz), 3.68 (3H, s), 3.79 (3H, s), 3.87 (1H, m), 3.95 (1H, m), 4.09 (1H, m), 4.42 (2H, ABq, J=11.0 Hz), 4.59 (1H, d, J=11.0 Hz), 4.65 (1H, d, J=11.8 Hz), 4.98 (1H, s), 5.02 (1H, d, J=15.2 Hz), 5.41 (1H, s), 5.88 (1H, s), 5.91 (1H, dd, J=16.2, 7.7 Hz), 6.83 (2H, d, J=8.7 Hz), 7.16 (2H, d, J=8.7 Hz), 7.27-7.35 (6H, m), 9.44 (1H, d, J=7.7 Hz); ¹³C NMR (75 M Hz, CDCl₃) δ 14.1, 21.7, 22.6, 24.0, 24.6, 28.9, 29.0, 31.7, 32.6, 34.5, 36.2, 47.5, 51.3, 51.5, 55.4, 69.2, 71.3, 71.8, 74.4, 76.3, 102.7, 114.1, 118.2, 127.1, 127.7, 127.9, 128.7, 129.5, 130.7, 138.9, 151.6, 159.6, 166.6, 167.3, 172.1, 195.0.

To a solution of 308.1 (17 mg, 0.02 mmol) in 0.5 mL 1% aqueous CH₂Cl₂ was added solid 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 8 mg, 0.03 mmol) at rt. The mixture was stirred for 2 h, pipetted directly onto a column of silica gel, and the product eluted with 35% EtOAc/hexanes to provide the corresponding intermediate alcohol (11 mg, 85%) as a colorless oil. R_(f) (50% EtOAc/hexanes)=0.55; IR 3528, 2930, 2858, 1745, 1720, 1686 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.86 (3H, t, J=6.9 Hz), 1.13 (3H, s), 1.17 (3H, s), 1.25 (10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12 (2H, m), 2.35 (1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H, s), 3.45 (1H, m), 3.68 (3H, s), 3.82 (1H, s), 4.24 (1H, m), 4.54 (2H, ABq, J=11.2 Hz), 5.47 (1H, s), 4.98 (1H, s), 5.02 (1H, d, J=15.2 Hz), 5.86 (1H, s), 5.91 (1H, dd, J=15.9, 7.5 Hz), 7.29 (1H, d, J=15.9 Hz), 7.34 (5H, s), 9.52 (1H, d, J=7.5 Hz); ¹³C-NMR (75 M Hz, CDCl₃) δ 13.8, 15.4, 21.7, 22.4, 23.6, 24.4, 28.8, 28.8, 31.5, 32.8, 34.4, 39.5, 47.4, 51.1, 51.2, 68.3, 70.8, 71.1, 71.2, 78.3, 102.5, 117.3, 127.0, 127.9, 128.0, 128.5, 138.1, 151.7, 166.5, 167.4, 171.8, 194.6.

The intermediate alcohol (11 mg, 0.02 mmol) was dissolved in 1.0 mL CH₃CN/H₂O (9:1) and treated with 48% aqueous HF (200 μL, 300 mol % excess) at rt. The resulting mixture was stirred for 1 h, quenched with sat. NaHCO₃ and diluted with 10 mL EtOAc. The aqueous layer was separated and extracted with EtOAc (2×). The combined organics were dried over Na₂SO₄ and concentrated in vacuo to afford the corresponding crude hemiketal enal of Formula 303a as a colorless oil. The crude product was purified by column chromatography on silica gel with 50% EtOAc-hexanes as eluant affording 8 mg (80%) of the C20 heptenoate hemiketal enal. R_(f) (35% EtOAc/hexanes)=0.05; IR 3528, 2930, 2858, 1745, 1720, 1686, 1458, 1437, 1380 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.86 (3H, t, J=6.9 Hz), 1.13 (3H, s), 1.17 (3H, s), 1.25 (10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12 (2H, m), 2.35 (1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H, s), 3.45 (1H, m), 3.68 (3H, s), 3.82 (1H, s), 4.24 (1H, m), 4.52 (2H, ABq, J=11.4 Hz), 4.98 (1H, s), 5.02 (1H, d, J=15.2 Hz), 5.47 (1H, s), 5.86 (1H, s), 5.91 (1H, dd, J=15.9, 7.5 Hz), 7.29 (1H, d, J=15.9 Hz), 7.34 (5H, s), 9.52 (1H, d, J=7.5 Hz); ¹³C-NMR (75 M Hz, CDCl₃) δ 13.9, 15.4, 21.7, 22.4, 23.6, 24.4, 28.8, 28.8, 31.4, 32.7, 34.2, 39.5, 47.4, 51.1, 51.2, 68.3, 70.9, 71.1, 71.1, 78.5, 102.4, 117.4, 126.8, 128.0, 128.0, 128.6, 138.1, 151.8, 166.4, 167.1, 171.8, 194.7.

Carboxylic acid 407 (21 mg, 0.04 mmol) and Et₃N (19 mL, 0.12 mmol) were dissolved in 400 μL toluene and treated with 2,4,6-trichlorobenzoylchloride (6.0 mL, 0.04 mmol) dropwise at rt. After 1 h at rt, a toluene solution of freshly prepared C20 heptenoate hemiketal enal (16 mg, 0.03 mmol) and 4-dimethylaminopyridine (17 mg, 0.13 mmol) was added gradually and stirring was continued for 40 min. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 20% EtOAc/hexanes to provide the expected ester (Formula 701 where R is OH, R′ is OBn, R³ is TBSO, R²⁰ is heptenoate, R²¹ is ═CH—CO₂Me and R²⁶ is methyl) as a colorless oil (24 mg, 80%). IR 3487, 2927, 2856, 1723, 1689, 1455, 1379 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.80 (1H, t, J=12.7 Hz), 0.93 (3H, t, J=7.0 Hz), 0.97 (3H, d, J=6.6 Hz), 1.10-1.36 (24H, m), 1.36-1.85 (21H, m), 1.91-2.13 (6H, m), 2.39 (1H, t, J=12.8 Hz), 2.79 (1H, d, J=13.2 Hz), 2.93 (1H, m), 3.05 (1H, m), 3.29 (3H, s), 3.33 (1H, s), 3.37-3.48 (2H, m), 3.80 (1H, dd, J=11.4, 5.1 Hz), 3.95-4.05 (2H, m), 4.15 (1H, td, J=10.8, 0.9 Hz), 4.23 (1H, d, J=13.5 Hz), 4.50 (2H, ABq, J=12.0 Hz), 4.98 (1H, s), 5.00 (1H, d, J=15.2 Hz), 5.57 (1H, s), 5.64 (1H, dd, J=10.6, 4.6 Hz), 6.05 (1H, dd, J=16.1, 7.6 Hz), 6.39 (1H, s), 7.10-7.35 (5H, m), 7.45 (1H, d, J=16.1 Hz), 9.60 (1H, d, J=7.6 Hz); ¹³C-NMR (75 M Hz, CDCl₃) δ 14.1, 15.1, 19.3, 20.1, 21.3, 22.3, 22.4, 22.7, 23.8, 24.0, 24.6, 24.6, 29.0, 29.0, 29.3, 31.3, 31.6, 31.8, 34.2, 34.5, 35.2, 35.7, 36.2, 37.6, 43.5, 45.7, 51.6, 59.1, 64.9, 66.7, 71.1, 72.0, 72.9, 74.1, 75.3, 77.1, 100.2, 100.6, 121.2, 127.2, 127.3, 127.9, 128.6, 138.9, 151.2, 164.5, 166.3, 171.5, 175.1, 193.4; [α]²⁰ _(D)=−19° (c 1.5, CH₂Cl₂).

To the ester prepared in the preceding step (21 mg, 0.03 mmol) in THF (0.5 mL) was added pyridine (360 mL, 0.45 mmol) followed by 70% HF/pyridine (144 μL, 500 mol % excess) and stirred for 20 hours. The reaction was then quenched with a saturated solution of sodium bicarbonate. The biphasic mixture was extracted with ethyl acetate (×4) and the combined organics were dried over sodium sulfate. The solvent was removed in vacuo to provide the corresponding crude C3 hydroxyester. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 30% EtOAc/hexanes to provide this corresponding ester (where R³ is OH) as a colorless oil (13 mg, 68%). R_(f) (30% EtOAc/hexanes)=0.23; IR 3522, 2927, 2857, 1724, 1664, 1230, 1158, 1136, 1107, 979 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.84 (3H, t, J=5.4 Hz), 0.88-0.96 (5H, m), 1.00 (3H, d, J=4.8 Hz), 1.02-1.55 (27H, m), 1.63-1.81 (2H, m), 1.82-1.94 (2H, m), 2.03 (1H, br t, J=5.2 Hz), 2.19-2.27 (1H, m), 2.34 (1H, dt, J=9, 1.5 Hz), 2.94-3.01 (2H, m), 3.22 (1H, s), 3.58 (1H, br d, J=3.6 Hz), 3.68-3.74 (1H, m), 3.84-3.88 (1H, m), 3.94 (1H, dd, J=8.6, 3.1 Hz), 4.23 (1H, dd, J=10.4, 1.7 Hz), 4.31 (1H, brt, J=8.1 Hz), 4.97 (1H, s), 5.02 (1H, d, J=15.2 Hz), 5.36-5.41 (1H, m), 5.50 (1H, s), 5.61 (1H, d, J=5.4 Hz), 6.00 (1H, dd, J=12.0, 5.4 Hz), 6.36 (1H, s), 6.53 (1H, d, J=12.0 Hz); ¹³C NMR (100 M Hz, CDCl₃) δ 14.2, 19.4, 19.8, 22.4, 22.9, 24.0, 24.5, 25.0, 29.1, 29.2, 30.2, 31.8, 31.9, 32.0, 33.1, 34.6, 34.9, 35.4, 36.5, 43.6, 45.6, 50.7, 66.1, 66.7, 69.6, 73.2, 74.7, 75.8, 76.3, 77.5, 98.7, 102.6, 120.5, 140.1, 151.2, 151.5, 166.5, 171.5, 174.2; [α]²⁰ _(D)=−130.5° (c 0.9, CDCl₃).

To a solution of the C3 hydroxy ester of the preceding step (12 mg, 0.01 mmol) in 1.0 mL CH₂Cl₂ was added 4 Å molecular sieves and the mixture was allowed to stand for 20 min. 45-50 beads of Amberlyst-15 sulfonic acid resin were added and the mixture was stirred at rt for 2 h. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 35% EtOAc/hexanes to provide the corresponding heptenoate macrocycle as a colorless oil (7 mg, 70%). R_(f) (35% EtOAc/hexanes)=0.21; IR 3522, 2927, 2857, 1724, 1664, 1230, 1158, 1136, 1107, 979 cm⁻¹; ¹H NMR (400 M Hz, CDCl₃) δ 0.84 (3H, t, J=5.4 Hz), 0.88-0.96 (5H, m), 1.00 (3H, d, J=4.8 Hz), 1.02-1.55 (27H, m), 1.63-1.81 (2H, m), 1.82-1.94 (2H, m), 2.03 (1H, br t, J=5.2 Hz), 2.19-2.27 (1H, m), 2.34 (1H, dt, J=9, 1.5 Hz), 2.94-3.01 (2H, m), 3.22 (1H, s), 3.58 (1H, br d, J=3.6 Hz), 3.68-3.74 (1H, m), 3.84-3.88 (1H, m), 3.94 (1H, dd, J=8.6, 3.1 Hz), 4.23 (1H, dd, J=10.4, 1.7 Hz), 4.31 (1H, brt, J=8.1 Hz), 4.99 (1H, s), 5.03 (1H, d, J=15.2 Hz), 5.36-5.41 (1H, m), 5.50 (1H, s), 5.61 (1H, d, J=5.4 Hz), 6.00 (1H, dd, J=12.0, 5.4 Hz), 6.36 (1H, s), 6.53 (1H, d, J=12.0 Hz); ¹³C NMR (100 M Hz, CDCl₃) δ 14.2, 19.4, 19.8, 22.4, 22.9, 24.0, 24.5, 25.0, 29.1, 29.2, 30.2, 31.8, 31.9, 32.0, 33.1, 34.6, 34.9, 35.4, 36.5, 43.6, 45.6, 50.7, 66.1, 66.7, 69.6, 73.2, 74.7, 75.8, 76.3, 77.5, 98.7, 102.6, 120.5, 140.1, 151.2, 151.5, 166.5, 171.5, 174.2.

The crude macrocycle of the preceding step (2 mg, 0.01 mmol) was dissolved in 0.5 mL EtOAc and 2.2 mg Pd(OH)₂ (20% wt. on carbon) was added. The resulting suspension was vigorously stirred under balloon pressure of hydrogen gas for 35 min. The crude mixture was pipetted directly onto a column of silica gel and the product was eluted with 60% EtOAc/hexanes to afford heptanoate analogue (702.3) (Formula II where R³ is OH, R²⁰ is —O—CO—C₆H₁₃, R²¹ is ═CH—CO₂Me, R²⁶ is methyl and X is oxygen) (1 mg, 63%) as a white semi-solid. R_(f) (50% EtOAc/hexanes)=0.21; ¹H NMR (300 M Hz, CDCl₃) δ 0.37 (3H, br. s), 0.79-0.92 (14H, m), 1.06 (3H, d, J=6.4 Hz), 1.07 (1H, t, J=11.0 Hz), 1.10-1.25 (5H, m), 1.27 (3H, s), 1.50 (3H, s), 1.57-1.78 (4H, m), 2.03 (2H, t, J=7.4 Hz), 2.17 (1H, dd, J=9.9/0.5 Hz), 2.37 (1H, m), 2.40 (1H, m), 2.85 (1H, t, J=11.2 Hz), 2.96 (1H, t, J=10.8 Hz), 3.15 (3H, s), 3.68-3.72 (3H, m), 3.91 (1H, dd, J=11.2, 4.0 Hz), 4.13 (1H, t, J=9.7 Hz), 4.35 (1H, dd, J=13.9, 2.2 Hz), 4.48 (1H, td, J=11.0/2.8 Hz), 4.70 (1H, d, J=12.1 Hz), 5.44 (1H, quint., J=4.8 Hz), 5.54 (1H, d, J=7.3 Hz), 5.69 (1H, s), 5.76 (1H, s), 5.85 (1H, dd, J=16.1, 7.5 Hz), 6.40 (1H, d, J=1.8 Hz), 6.50 (1H, d, J=15.9 Hz); ¹³C NMR (125 M Hz, CDCl₃) δ 14.2, 19.4, 19.8, 22.4, 22.9, 24.0, 24.5, 25.0, 29.1, 29.2, 30.2, 31.8, 31.9, 32.0, 33.1, 34.6, 34.9, 35.4, 36.5, 43.6, 45.6, 50.7, 66.1, 66.7, 69.6, 73.2, 74.7, 75.8, 76.3, 77.5, 98.7, 102.6, 120.5, 140.1, 151.2, 151.5, 166.5, 171.5, 174.2.

4C. Myristate C20 Ester (702.4)

To solution of the enal of Formula 305 (in which R²⁰ is C₇H₁₅) (180 mg, 0.03 mmol), in 0.5 mL of MeOH at rt was added PPTS (2 mg, catalytic) and trimethylorthoformate (5 drops). The progress of the reaction was monitored by TLC. After 30 min the reaction was quenched with 1.0 mL Et₃N. The solvent was removed under reduced pressure to afford the corresponding crude dimethylacetal according to Formula 306. The dimethylacetyl was immediately dissolved in MeOH (0.5 mL) and K₂CO₃ (3 mg, catalytic). The progress of the reaction was monitored by TLC. After 30 min the reaction was quenched. The solution was quenched with sat. NaHCO₃, diluted with EtOAc (10 mL), washed with H₂O, dried over MgSO₄ and concentrated under reduced pressure to afford crude C20 free hydroxyl product of Formula 307, which was reacted with myristic acid in the same manner as the reaction of heptenoic acid in Example 4B. After 30 min the reaction was quenched. The solution was quenched with sat. NaHCO₃, diluted with EtOAc (10 mL), washed with H₂O, dried over MgSO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel with 35% EtOAc-hexanes as eluant affording 14 mg (70% for three steps) of the desired dimethylacetal myristate of Formula 308. R_(f) (20% ethyl acetate/hexanes)=0.5; R_(f) (35% EtOAc/hexanes)=0.50; IR 2927, 2859, 1744, 1719, 1687, 1514, 1249, 1156, 1103, 1079, 1037 cm⁻¹; ¹HNMR (300 M Hz, CDCl₃) δ 0.85 (6H, m) 1.16-1.30 (10H, m), 1.14 (3H, s), 1.18 (3H, s), 1.75 (1H, m), 1.95-2.17 (3H, m), 2.32 (1H, m), 3.37 (3H, s), 3.52 (1H, d, J=16.5 Hz), 3.68 (3H, s), 3.79 (3H, s), 3.87 (1H, m), 3.95 (1H, m), 4.09 (1H, m), 4.55 (2H, ABq, J=11.0 Hz), 5.41 (1H, s), 5.86 (1H, s), 5.91 (1H, dd, J=16.2, 7.6 Hz), 6.83 (2H, d, J=8.7 Hz), 7.16 (2H, d, J=8.7 Hz), 7.30-7.35 (6H, m), 9.43 (1H, d, J=7.6 Hz); ¹³C NMR (75 M Hz, CDCl₃) δ 14.1, 21.7, 22.6, 24.0, 24.6, 28.9, 29.0, 31.7, 32.6, 34.5, 36.2, 47.5, 51.3, 51.5, 55.4, 69.2, 71.3, 71.8, 74.4, 76.3, 102.7, 114.1, 118.2, 127.1, 127.7, 127.9, 128.7, 129.5, 130.7, 138.9, 151.6, 159.6, 166.6, 167.3, 172.1, 195.0.

To a solution of the dimethylacetal myristate (11 mg, 0.01 mmol) in 0.6 mL 1% aqueous CH₂Cl₂ was added solid 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 4 mg, 0.02 mmol) at rt. The mixture was stirred for 2 h, pipetted directly onto a column of silica gel, and the product eluted with 35% EtOAc/hexanes to provide the corresponding intermediate alcohol (8 mg, 89%) as a colorless oil: R_(f) (35% EtOAc/hexanes)=0.22; IR 3528, 2930, 2858, 1745, 1720, 1686, 1458, 1437, 1380 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.86 (3H, t, J=6.9 Hz), 1.13 (3H, s), 1.17 (3H), 1.25 (10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12 (2H, m), 2.35 (1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H, s), 3.45 (1H, m), 3.68 (3H, s), 3.82 (1H, s), 4.24 (1H, m), 4.59 (2H, ABq, J=11.4 Hz), 5.47 (1H, s, C20), 5.86 (1H, s), 5.91 (1H, dd, J=15.9, 7.5 Hz), 7.29 (1H, d, J=15.9 Hz), 7.34 (5H, m), 9.52 (1H, d, J=7.5 Hz); ¹³C-NMR (75 M Hz, CDCl₃) δ 13.86, 15.44, 21.70, 22.38, 23.61, 24.39, 28.75, 28.81, 31.45, 32.75, 34.22, 39.50, 47.35, 51.13, 51.23, 68.32, 70.89, 71.06, 71.12, 78.51, 102.36, 117.43, 126.83, 127.97, 127.98, 128.57, 138.11, 151.80, 166.42, 167.11, 171.76, 194.73; [α]_(D) ²⁰=−21.0° (c 1.0, CH₂Cl₂).

The intermediate alcohol (7 mg, 0.02 mmol) was dissolved in 1.1 mL CH₃CN/H₂O (9:1) and treated with 48% aqueous HF (200 μl, 300 mol % excess) at rt. The resulting mixture was stirred for 1 h, quenched with sat. NaHCO₃ and diluted with 10 mL EtOAc. The aqueous layer was separated and extracted with EtOAc (2×). The combined organics were dried over Na₂SO₄ and concentrated in vacuo to afford the crude hemi-ketal enal (44 in Reaction Scheme 11, which provides the compound number references for the remainder of the present example) as a colorless oil. The crude product was purified by column chromatography on silica gel with 35% EtOAc-hexanes as eluant affording 6 mg (86%) of enal 44. R_(f) (35% EtOAc/hexanes)=0.15; IR 3528, 2930, 2858, 1745, 1720, 1686, 1458, 1437, 1380 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.87 (3H, t, J=6.9 Hz), 1.13 (3H, s), 1.15 (3H, s), 1.28 (10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12 (2H, m), 2.35 (1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H, s), 3.45 (1H, m), 3.68 (3H, s), 3.82 (1H, s), 4.24 (1H, m), 4.56 (2H, ABq, J=11.0 Hz), 5.47 (1H, s), 5.86 (1H, s), 5.91 (1H, dd, J=15.9, 7.4 Hz), 7.28 (1H, d, J=15.9 Hz), 7.35 (5H, s), 9.52 (1H, d, J=7.4 Hz); ¹³C-NMR (75 M Hz, CDCl₃) δ 13.9, 14.1, 19.4, 20.0, 22.7, 23.0, 24.4, 24.6, 29.1, 29.1, 29.4, 29.4, 29.6, 29.7, 31.2, 31.4, 31.9, 32.4, 34.6, 36.0, 40.0, 42.6, 42.9, 45.0, 51.1, 64.5, 66.3, 68.7, 70.4, 73.7, 74.1, 75.8, 76.1, 77.2, 78.7, 94.0, 98.9, 102.4, 119.7, 125.7, 142.8, 151.8, 167.0, 172.1, 172.5, 194.7.

Carboxylic acid 6 (6 mg, 0.01 mmol) and Et₃N (6 μL, 0.04 mmol) were dissolved in 300 μL toluene and treated with 2,4,6-trichlorobenzoylchloride (2.0 μL, 0.01 mmol) dropwise at rt. After 1 h at rt, a toluene solution of freshly prepared enal 44 and 4-dimethylaminopyridine (5 mg, 0.04 mmol) was added gradually and stirring was continued for 40 min. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 20% EtOAc/hexanes to provide ester-enal 46 as a colorless oil (9 mg, 90%). R_(f) (35% EtOAc/hexanes)=0.71; IR 3487, 2927, 2856, 1723, 1689, 1455, 1379, 1228, 1156, 1113, 1084, 1032, 981 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.80 (1H, t, J=12.7 Hz), 0.93 (3H, t, J=7.0 Hz), 0.98 (3H, d, J=6.6 Hz), 1.10-1.41 (24H, m), 1.42-1.85 (21H, m), 1.92-2.13 (6H, m), 2.39 (1H, t, J=12.7 Hz), 2.81 (1H, d, J=13.2 Hz), 2.93 (1H, m), 3.05 (1H, m), 3.29 (3H, s), 3.33 (1H, s), 3.37-3.48 (2H, m), 3.80 (1H, dd, J=11.4, 5.1 Hz), 3.95-4.05 (2H, m), 4.15 (1H, td, J=10.8, 0.9 Hz), 4.23 (1H, d, J=13.5 Hz), 4.46 (2H, ABq, J=11.0 Hz), 5.57 (1H, s), 5.64 (1H, dd, J=10.6, 4.6 Hz), 6.05 (1H, dd, J=15.9, 7.5 Hz), 6.39 (1H, s), 7.10-7.35 (5H, m), 7.45 (1H, d, J=15.9 Hz), 9.60 (1H, d, J=7.5 Hz); ¹³C-NMR (75 M Hz, CDCl₃) δ −9.6, −9.4, 9.2, 10.3, 13.2, 14.1, 15.1, 19.3, 20.1, 21.3, 22.3, 22.4, 22.7, 23.8, 24.0, 24.6, 24.6, 29.0, 29.0, 29.3, 31.3, 31.6, 31.8, 34.2, 34.5, 35.2, 35.7, 36.2, 37.6, 43.5, 45.7, 51.6, 59.1, 64.9, 66.7, 71.1, 72.0, 72.9, 74.1, 75.3, 77.1, 100.2, 100.6, 121.2, 127.2, 127.3, 127.9, 128.6, 138.9, 151.2, 164.5, 166.3, 171.5, 175.1, 193.2; [α]²⁰ _(D)=−19° (c 1.5, CH₂Cl₂).

To ester-enal 46 (8.0 mg, 0.001 mmol) in THF (0.5 mL) was added 70% HF/pyridine (0.3 mL, 0.3 mmol) and stirred for 2 hours. The reaction was then quenched with a saturated solution of sodium bicarbonate. The biphasic mixture was extracted with ethyl acetate (×4) and the combined organics were dried over sodium sulfate. The solvent was removed in vacuo to provide crude macrocycle. The crude mixture was chromatographed on silica gel and the product was eluted with 50% EtOAc/hexanes to afford 5.0 mg (83%) of the corresponding macrocycle as an clear oil.: R_(f) (40% EtOAc/hexanes)=0.19; IR 3522, 2927, 2857, 1724, 1664, 1230, 1158, 1136, 1107, 979 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.84 (3H, t, J=5.4 Hz), 0.88-0.96 (5H, m), 1.00 (3H, d, J=4.8 Hz), 1.02-1.55 (27H, m), 1.63-1.81 (2H, m), 1.82-1.94 (2H, m), 2.03 (1H, br t, J=5.2 Hz), 2.19-2.27 (1H, m), 2.34 (1H, dt, J=9, 1.5 Hz), 2.94-3.01 (2H, m), 3.22 (1H, s), 3.58 (1H, br d, J=3.6 Hz), 3.68-3.74 (1H, m), 3.84-3.88 (1H, m), 3.94 (1H, dd, J=8.6, 3.1 Hz), 4.23 (1H, dd, J=10.4, 1.7 Hz), 4.31 (1H, br t, J=8.1 Hz), 4.56 (2H, ABq, J=11.0 Hz), 5.36-5.41 (1H, m), 5.50 (1H, s), 5.61 (1H, d, J=5.4 Hz), 6.00 (1H, dd, J=12.0, 5.4 Hz), 6.36 (1H, s), 6.53 (1H, d, J=12.0 Hz); ¹³C NMR (100 M Hz, CDCl₃) δδ 14.1, 19.4, 20.0, 22.7, 23.0, 24.4, 24.6, 29.1, 29.1, 29.4, 29.4, 29.6, 29.7, 31.2, 31.4, 31.9, 32.4, 34.6, 36.0, 40.0, 42.6, 42.9, 45.0, 51.1, 64.5, 66.3, 68.7, 70.4, 73.7, 74.1, 75.8, 76.1, 77.2, 78.7, 94.0, 98.9, 102.4, 119.7, 125.7, 142.8, 151.8, 167.0, 172.1, 172.5.

To 5.0 mg (0.0005 mmol) of crude macrocycle of the preceding step in ethyl acetate (1.0 ml) was added a catalytic amount of Pearlman's catalyst. The flask was evacuated and refilled with a 1 atm. hydrogen atmosphere (×4), stirred under hydrogen for 30 min, and then pipetted directly onto a silica gel column and eluted with 60% ethyl acetate/hexanes. This process afforded 4.8 mg (99%) of analogue 48 (702.4) (Formula II where R³ is OH, R²⁰ is —O—CO—C₁₃H₂₇, R²¹ is ═CH—CO₂Me, R²⁶ is methyl and X is oxygen) as an amorphous solid. R_(f) (50% EtOAc/hexanes)=0.16; IR 3522, 2927, 2857, 1724, 1664, 1230 IR (neat)=3455, 3319, 2929, 2856, 1735, 1380, 1230, 1138, 976 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.37 (3H, bs), 0.79-0.92 (14H, m), 1.06 (3H, d, J=6.4 Hz), 1.07 (1H, t, J=11.0 Hz), 1.10-1.25 (5H, m), 1.27 (3H, s), 1.50 (3H, s), 1.57-1.78 (4H, m), 2.03 (2H, t, J=7.4 Hz), 2.17 (1H, dd, J=9.9/0.5 Hz), 2.37 (1H, m), 2.40 (1H, m), 2.85 (1H, t, J=11.2 Hz), 2.96 (1H, t, J=10.8 Hz), 3.15 (3H, s), 3.68-3.72 (3H, m), 3.91 (1H, dd, J=11.2, 4.0 Hz), 4.13 (1H, t, J=9.7 Hz), 4.35 (1H, dd, J=13.9/2.2 Hz), 4.48 (1H, td, J=11.0, 2.8 Hz), 4.70 (1H, d, J=12.1 Hz), 5.44 (1H, quint., J=4.8 Hz), 5.54 (1H, d, J=7.3 Hz), 5.69 (1H, s), 5.76 (1H, s), 5.85 (1H, dd, J=16.1, 7.5 Hz), 6.40 (1H, d, J=1.8 Hz), 6.50 (1H, d, J=15.9 Hz); ¹³C NMR (125 M Hz, CDCl₃) δ 14.1, 19.4, 20.0, 22.7, 23.0, 24.4, 24.6, 29.1, 29.1, 29.4, 29.4, 29.6, 29.7, 31.2, 31.4, 31.9, 32.4, 34.6, 36.0, 40.0, 42.6, 42.9, 45.0, 51.1, 64.5, 66.3, 68.7, 70.4, 73.7, 74.1, 75.8, 76.1, 77.2, 78.7, 94.0, 98.9, 102.4, 119.7, 125.7, 142.8, 151.8, 167.0, 172.1, 172.5.

4D. Benzoate C20 Ester (702.5)

Enal 45 was prepared following the procedure for compound III in Example IC except that benzoic acid was substituted for octanoic acid, to form the corresponding protected benzoate product.

Carboxylic acid 6 (6 mg, 0.01 mmol) and Et₃N (6 μL, 0.04 mmol) were dissolved in 300 μL toluene and treated with 2,4,6-trichlorobenzoylchloride (2 μL, 0.01 mmol) dropwise at rt. After 1 h at rt, a toluene solution of freshly prepared 45 and 4-dimethylaminopyridine (5 mg, 0.01 mmol) was added gradually and stirring was continued for 40 min. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 20% EtOAc/hexanes to provide the expected ester product as a colorless oil (8 mg, 89%). R_(f) (35% EtOAc/hexanes)=0.71; IR 3460, 2927, 2856, 1723 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.08 (3H, s), 0.08 (3H, s), 0.81 (12H, m), 0.82-0.96 (3H, m), 1.06-1.32 (15H, m), 1.59-1.81 (4H, m), 2.20 (1H, t, J=9.3 Hz), 3.33-3.44 (2H, m), 3.67-3.84 (1H, m), 4.00-4.15 (4H, m), 4.31-4.39 (1H, m), 4.55 (2H, ABq, J=8.5 Hz), 5.41 (1H, s), 5.75 (1H, dd, J=15.5, 3.4 Hz), 6.01 (1H, s), 6.39 (1H, s), 7.28-7.47 (9H, m), 7.84 (1H, d, J=6.9 Hz), 9.17 (1H, d, J=7.3 Hz); ¹³C-NMR (75 M Hz, CDCl₃) δ −9.6, −9.4, 9.2, 10.3, 13.2, 14.2, 18.5, 22.0, 23.5, 28.4, 28.7, 30.7, 31.5, 33.5, 39.1, 41.5, 41.9, 44.0, 50.1, 63.7, 65.3, 67.8, 69.6, 70.3, 73.8, 74.8, 75.1, 76.2, 77.7, 98.1, 101.3, 118.8, 124.7, 126.7, 127.4, 127.5, 128.9, 132.2, 137.3, 141.8, 150.8, 163.6, 165.9, 170.2.

To the ester of the preceding step (13 mg, 0.02 mmol) in THF (0.5 mL) was added pyridine (360 μL, 0.45 mmol) followed by 70% HF/pyridine (144 μL, 500 mol % excess) with stirring for 20 hours. The reaction was then quenched with a saturated solution of sodium bicarbonate. The biphasic mixture was extracted with ethyl acetate (×4) and the combined organics were dried over sodium sulfate. The solvent was removed in vacuo to provide the corresponding crude C3 hydroxyester. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 35% EtOAc/hexanes to provide the purified C3 hydroxyester as a colorless oil (9 mg, 82%). R_(f) (40% EtOAc/hexanes)=0.19; IR 3522, 2927, 2857, 1724 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) 0.81-0.91 (3H, m), 1.08 (3H, s), 1.20-1.59 (9H, m), 1.31 (6H, s), 1.92-2.18 (4H, m), 2.45 (2H, bs), 3.40-3.58 (2H, m), 3.67 (3H, s), 3.69-3.78 (2H, m), 3.88-3.98 (2H, m) 4.03-4.24 (3H, m), 4.45 (1H, d, J=9.2 Hz), 4.65 (2H, ABq, J=8.5 Hz), 5.17 (1H, d, J=9.7 Hz), 5.22 (1H, s), 5.40 (1H, s), 5.44 (1H, dd, J=15.5, 7.3 Hz), 6.06 (1H, s), 6.08 (1H, d, J=15.5 Hz), 7.27-7.59 (9H, m), 8.05 (1H, d, J=6.9 Hz); ¹³C NMR (100 M Hz, CDCl₃) δ 14.2, 18.5, 22.0, 23.5, 28.4, 28.7, 30.7, 31.5, 33.5, 39.1, 41.5, 41.9, 44.0, 50.1, 63.7, 65.3, 67.8, 69.6, 70.3, 73.8, 74.8, 75.1, 76.2, 77.7, 98.1, 101.3, 118.8, 124.7, 126.7, 127.4, 127.5, 128.9, 132.2, 137.3, 141.8, 150.8, 163.6, 165.9, 170.2; [α]²⁰ _(D)=−110.5° (c 0.9, CDCl₃).

To 4.0 mg (0.001 mmol) of the crude C3 hydroxyester of the preceding step in ethyl acetate (1.0 ml) was added a catalytic amount of Pearlman's catalyst. The flask was evacuated and refilled with a 1 atm. hydrogen atmosphere (×4). Stirred under hydrogen for 30 min. and then pipetted directly onto a silica gel column and eluted with 60% ethyl acetate/hexanes. HPLC (hexane: methylene chloride: i-propanol, 16:3:1) Isolated 2.2 mg (63%) of analogue 49 (702.5) (Formula II where R³ is OH, R²⁰ is —O—CO-Ph, R²¹ is ═CH—CO₂Me, R²⁶ is methyl and X is oxygen) as an amorphous solid. R_(f) (50% EtOAc/hexanes)=0.16; IR 3522, 2927, 2857, 1724, 1664, 1230 IR (neat)=3455, 3319, 2929, 2856, 1735, 1380, 1230, 1138, 976 cm⁻¹; ¹H NMR (300 M Hz, CDCl₃) δ 0.81-0.92 (3H, m), 1.08 (3H, s), 1.20-1.59 (9H, m), 1.32 (6H, s), 1.90-2.18 (4H, m), 2.52-2.56 (2H, m), 3.40-3.55 (2H, m), 3.67 (3H, s), 3.73-3.79 (2H, m), 3.82-3.95 (2H, m) 4.02-4.22 (2H, m), 4.51 (1H, d, J=9.1 Hz), 5.11 (1H, d, J=8.9 Hz), 5.26 (1H, s), 5.40 (1H, s), 5.42 (1H, dd, J=15.5, 3.4 Hz), 6.04 (1H, d, J=15.5 Hz), 6.07 (1H, s), 7.34-7.57 (4H, m), 8.04 (1H, d, J=7.3 Hz); ¹³C NMR (125 M Hz, CDCl₃) δ 13.1, 18.4, 18.9, 21.7, 23.5, 28.7, 30.6, 31.4, 35.0, 39.1, 41.6, 50.1, 63.6, 67.7, 69.2, 72.6, 76.1, 77.7, 93.0, 99.2, 113.0, 124.7, 127.2, 127.5, 128.9, 132.2, 146.3, 148.1, 149.9, 150.6, 171.4, 173.2; [α]²⁵ _(D)=−70.0° (c=0.36, CDCl₃).

Example 5 Protein Kinase C (Isozyme Mix) Assay Protocol

The following procedure was used. Filters (Whatman GF-B, 21 mm diam.) are soaked for 1 h in a solution containing deionized water (97 mL), and 10% polyethyleneamine (3 mL). A filtering buffer solution containing TRIS (1M, pH 7.4, 10 mL) and water (490 mL) is prepared and cooled on ice. An assay buffer solution is prepared by the addition of TRIS (1M, pH 7.4, 1 mL), KCl (1M, 2 mL), CaCl₂ (0.1M, 30 μL), bovine serum albumin (40 mg), diluted to 20 mL with deionized water and stored on ice. Phosphatidyl serine vesicles are prepared by the addition of phosphatidyl serine (10 mg/mL in chloroform, 0.4 mL) to a glass test tube followed by removal of the chloroform under a stream of nitrogen (5 min). To this viscous liquid is added a portion of the prepared assay buffer (4 mL) and the resulting mixture is then transferred to a plastic tube with washing. This tube is then sonicated (Branson Sonifier 250, power=6, 40% duty cycle) four times for 30 sec. with a 30 sec. rest period between sonications. The resulting solution is stored over ice. PKC is prepared by addition of cooled assay buffer (10 mL) to PKC (25 μL) purified from rat brain and then stored on ice. Stock solutions of compounds are diluted with absolute ethanol in glass in serial fashion. Each plastic assay incubation tube is made to contain prepared phosphatidyl serine vesicles (60 μL), prepared PKC solution (200 μL) and analogue (0-20 μL) plus EtOH (20-0 μL) for a total volume of 20 μL). Lastly, tritiated phorbol 12,13-dibutyrate (PDBU) (30 nM, 20 μL) is added to each tube. The assay is carried out using 7-10 analogue concentrations, each in triplicate. Non-specific binding is measured in 1-3 tubes by the substitution of phorbol myristate acetate (PMA) (1 mM, 5 μL) and EtOH (15 μL) for the analogue/EtOH combination. The tubes are incubated at 37° C. for 90 min. and then put on ice for 5 min. Each tube is then filtered separately through a pre-soaked filter disc. Each tube is rinsed with cold 20 mM TRIS buffer (500 μL) and the rinseate is added to the filter. The filter is subsequently rinsed with cold 20 mM TRIS buffer (5 mL) dropwise. The filters are then put in separate scintillation vials and Universol© scintillation fluid is added (3 mL). The filters are immediately counted in a scintillation counter (Beckman LS 6000SC). Counts per minute are averaged among three trials at each concentration. The data is then plotted using a least squares fit algorithm with the Macintosh version of Kaleidagraph© (Abelbeck Software) and an IC₅₀ (defined as the concentration of analogue required to displace half of the specific PDBU binding to PKC) is calculated. The IC₅₀ then allows determination of the K_(i) for the analogue from the equation: K_(i)=IC₅₀/(1+[PDBu]/K_(d) of PDBu). The K_(d) of [H³]-PDBu was determined under identical conditions to be 1.55 nM.

Example 6 PKCδ-C1B Assay Protocol

All aspects of the PKCδ-C1B assay are identical to the PKC isozyme mix assay from Example 5 except the following features: In the PKCδ-C1B assay system, assay buffer is made without CaCl₂. PKCδ-C1B (200 μg, 34.14 nmol), is dissolved in deionized water (160 μL) and ZnCl₂ (5 mM, 40 μL) is added. The resulting solution is allowed to stand at 4° C. for 10 min. An aliquot (10 μL) of this solution is diluted to 2 mL with deionized water. An aliquot (290 μL) is further diluted to 20 mL with assay buffer and is ready for use. The incubation time is shortened from 90 min. to 30 min. Lastly, during the filtering portion of the assay, the tube is not washed with filtering buffer (0.5 mL).

When tested as described above, the C26 desmethyl analogue 702.1 (Example 3A), had significantly higher activity than the corresponding C26 methyl-containing analogue (Formula 1998a where R³ is OH). Similarly, among several analogues having different C20 ester groups, the presence of longer R²⁰ substituents (48) or an aryl substituent (49) also afforded higher activity. The results are shown below in Table 1 (in all compounds tested, R³ was OH and R²¹ was ═CH—CO₂Me).

TABLE 1 PKCδ-C1B Assay Compound R²⁰ R²⁶ Ki (nM) Phorbol 1.7 (K_(d) dibutyrate value) Formula 1998a —OC(O)C₇H₁₅ CH₃ 5.1 Formula IIa —OC(O)C₇H₁₅ H 0.30 ± 0.07 (702.1) Formula 702.2 —OC(O)CH₃ CH₃ 232 ± 11  Formula 702.3 —OC(O)C₆H₁₃ CH₃ 35 Formula 702.4 —OC(O)C₁₃H₂₇ CH₃ 1.3 Formula 702.5 —OC(O)Phenyl CH₃ 1.7

Example 7 P388 Murine Lymphocytic Leukemia Cell Assay

Cells from a P388 cell line (CellGate, Inc., Sunnyvale, Calif.) are grown in RPMI 1640 cell medium containing fetal calf serum (10%), L-glutamine, penicillin, streptomycin and are split twice weekly. All compounds are first diluted with DMSO. Later serial dilutions are done with a phosphate buffer solution (HYQ DPBS modified phosphate buffered saline). All dilutions are done in glass vials and the final DMSO concentration is always below 0.5% by volume. Final two-fold dilutions are done in a 96 well plate using cell media so that each well contains 50 μL. All compounds are assayed in quadruplicate over 12 concentrations. Cell concentration is measured using a hemacytometer and the final cell concentration is adjusted to 1×10⁴ cells/mL with cell medium. The resulting solution of cells (50 μL) is then added to each well and the plates are incubated for 5 days in a 37° C., 5% CO₂, humidified incubator (Sanyo CO₂ incubator). MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 10 μL) is then added to each well and the plates are re-incubated under identical conditions for 2 h. To each well is then added acidified isopropanol (150 μL of i-PrOH solution containing 0.05 N HCl) and mixed thoroughly. The plates are then scanned at 595 nm and the absorbances are read (Wallac Victor 1420 Multilabel Counter). The resulting data is then analyzed to determine an ED₅₀ value using the Prism software package (GraphPad).

When tested as described above, the C26 desmethyl analogue 702.1 (Example 3A), had significantly higher activity than the corresponding C26 methyl-containing analogue. Similarly, among several analogues having different C20 ester groups, the presence of longer R20 substituents (48) or an aryl substituent (49) also afforded higher activity. The results are shown below in Table 2 (in all compounds tested, R³ was OH and R²¹ was ═CH—CO₂Me).

TABLE 2 P388 Assay Compound R²⁰ R²⁶ ED₅₀ (nM) Formula 1998a —OC(O)C₇H₁₅ CH₃ 76 Formula IIa —OC(O)C₇H₁₅ H 17 (702.1) Formula 702.2 —OC(O)CH₃ CH₃ 181 Formula 702.3 —OC(O)C₆H₁₃ CH₃ 38 Formula 702.4 —OC(O)C₁₃H₂₇ CH₃ 3.6 Formula 702.5 —OC(O)Phenyl CH₃ 42

Example 8 In Vitro Inhibition of Growth in Human Cancer Cell Lines

Anticancer data were obtained in vitro for C26 desmethyl bryostatin analogue 702.1 (Example 3A) tested against a spectrum of different NCl human cancer cell-lines associated with various cancer conditions. The results are shown in Table 3. Data obtained with Bryostatin-1 are included for comparison. Growth inhibition (GI50) values are expressed as the log of molar concentration at half-maximum inhibition. As can be seen, the C26 desmethyl compound was at least as potent, on average, as bryostatin-1 for all cell groups tested. Moreover, the C26 desmethyl compound was more active than bryostatin-1 by more than 2 orders of magnitude for several cell lines: K-562 and MOLT-4 (leukemia), NCI-H460 (NSC lung), HCC-2998 (colon), TK-10 (renal), and MDA-MB-435 (breast). These results are significant and surprising since the C27 methyl group attached to C26 was previously believed to be necessary for activity.

TABLE 3 Cell Line: Desmethyl Bryo-1 Difference Leukemia CCRF-CEM −5.81 −5.30 −0.51 HL-60(TB) −5.84 −5.70 −0.14 K-562 −7.5 −5.40 −2.10 MOLT-4 <−8.0 −5.50 −2.50 RPMI-8226 −5.82 >−5 −0.82 SR −5.1 >−5 −0.10 NSC Lung A549/ATCC −6.62 −5.20 −1.42 EKVX −5.58 −5.30 −0.28 HOP-62 −4.79 >−5 HOP-92 −4.67 −5.30 0.63 NCI-H226 >−5 NCI-H23 >−5 NCI-H322M −4.38 −6.00 1.62 NCI-H460 <−8 −5.60 −2.40 NCI-H522 >−5 Colon COLO 205 −7.08 −5.40 −1.68 HCC-2998 −7.54 −5.30 −2.24 HCT-116 −5.32 −5.30 −0.02 HCT-15 −4.76 >−5 HT29 −5.55 −5.30 −0.25 KM12 −5.34 −5.20 −0.14 SW-620 −5.12 −5.50 0.38 CNS SF-268 −4.97 −5.10 0.13 SF-295 −6.05 −5.20 −0.85 SF-539 −5.77 >−5 −0.77 SNB-19 −5.2 >−5 −0.20 SNB-75 −4.97 −5.50 0.53 U251 −5.4 −5.10 −0.30 Prostate PC-3 −5.6 −5.30 −0.30 DU-145 −5.02 >−5 −0.02 Melanoma LOX IMVI −5.47 −5.10 −0.37 MALME-3M −5.20 MI4 −5.26 >−5 −0.26 SK-MEL-2 −5.02 −5.20 0.18 SK-MEL-28 −4.67 −5.10 0.43 SK-MEL-5 −6.43 −5.70 −0.73 UACC-257 −5.13 −5.10 −0.03 UACC-62 −5.11 −5.30 0.19 Ovarian IGROVI −4.88 −5.30 0.42 OVCAR-3 −4.83 −5.10 0.27 OVCAR-4 −5.15 −5.50 0.35 OVCAR-5 −5.28 >−5 −0.28 OVCAR-8 −4.77 −5.10 0.33 SK-OV-3 −4.3 −5.10 0.80 Renal 786-0 −5.46 −5.20 −0.26 A498 −6.38 >−5 −1.38 ACHN −5.94 −5.50 −0.44 CAKI-1 −5.7 −5.40 −0.30 RXF-393 −5.30 SN12C −5.59 −5.10 −0.49 TK-10 −7.03 >−5 −2.03 UO-31 −4.85 −5.60 0.75 Breast MCF7 −5.4 −5.20 −0.20 NCI/ADR-RES −4.74 >−5 MDA-MB-231/ATCC −5.69 −5.20 −0.49 MDA-MB-435 −7.66 −5.10 −2.56 MDA-N −5.10 BT-549 −4.71 −5.10 0.39 T-47D −5.02 −5.20 0.18 HS 578T −5.18 −5.2 0.02

Example 9 Formula 405 where X is —O—

In an oven dried argon purged 100 mL round-bottom flask charged with a magnetic stir bar was added a solution of 482 mg (1.49 mmol, 1.0 eq) of the compound of Formula 404.1 in 31 mL of isobutylvinyl ether. 237 mg (0.744 mmol, 0.5 eq) of Hg(II) diacetate was added in one portion at room temperature [rt]. The reaction was run at rt for 48 hrs. The reaction was diluted with EtOAc and washed with sat. NaHCO₃ and brine. The combined organic layers were dried over MgSO₄, filtered and concentrated. Rapid flash chromatography using 15% ethyl acetate, 85% petroleum ether plus 1% triethyl amine yielded crude vinylated pyran. This was carried on immediately.

In an oven-dried argon-purged 250 mL round-bottom flask charged with a magnetic stir bar was added a solution of 480 mg (1.37 mmol, 1.0 eq) of the vinylated pyran in 26 mL of degassed, (via bubbling argon gas through), 99%+anhydrous decane (Aldrich). The reaction vessel was lowered into a preheated 155° C. oil bath. The reaction was heated for 3 hrs at this temperature under an argon atmosphere. It was then removed from the oil bath and cooled to rt. It was allowed to sit overnight under an argon atmosphere. The whole reaction was loaded onto a column using petroleum ether to assist in the transfer. The column was then eluted with 10% ethyl acetate 90% petroleum ether, to yield 431 mg (83% over two steps) of the title compound of Formula 405, [6-(7-isopropyl-10-methyl-1,5-dioxa-spiro[5.5]undec-2-ylmethyl)-5,6-dihydro-2H-pyran-2-yl]-acetaldehyde, as a clear colorless oil.

Example 10

Procedure:

6.38 g of freshly distilled diisopropyl amine was combined with 100 ml of dry THF and cooled to −78° C. 40.7 mL of 1.55 M n-BuLi in Hexanes was then added slowly. After 10 minutes at −78° C., the solution was warmed to 0° C. for 15 minutes then re-cooled to −78° C. To this solution of lithium diisopropylamide [LDA] at −78° C. was added 5 g of tert-butylacetoacetate slowly. After 10 minutes at −78° C., the solution was warmed to 0° C. for 40 minutes. The bright yellow solution of dianion was then cooled to −78° C. and 2.97 g of diethylglutarate was added in 5 mL of dry THF in one portion. After 30 minutes at −78° C., the reaction was quenched by the addition of 140 mL of 1 N HCl in water. The reaction was then partitioned between the aqueous layer and 200 mL of ether. The ether was removed in vacuo and the residue purified via flash chromatography (25% Ether:Pentane —R_(f)=0.3) to give 65% product.

Procedure:

A 2-neck flask with 2 gas flow adaptors was charged with 21 mg of COD-RuBis-2-methylallyl complex and 50 mg of (S)-BINAP. To this was added 4 mL of degassed acetone (Argon purge for 20 min.) and 12 mg of HBr (24 μL of 49% soln) in 0.4 mL degassed MeOH (as above). The solution became red-orange with a red precipitate. After 30 minutes, the solvents were carefully remove in vacuo (air sensitive!) to yield a tan powder that was used directly. To the crude (S)-BINAP-RuBr₂ was added 1 g of 3,5-dioxo-nonanedioic acid 1-tert-butyl ester 9-ethyl ester in 10 mL EtOH. The solution was stirred rapidly and the suspension transferred to a Parr Bomb apparatus under Argon blanket. The bomb was sealed and pressurized to 20 atmospheres for 3 cycles then left at 20 atmospheres and heated to 75° C. with stirring. After 6 hours, the reaction appeared complete by TLC and the product isolated by Flash Chromatography (R_(f)=0.2 in 1:1 Ethyl Acetate:Pentane) to yield 442 mg of product that was recrystallized in Ethyl Acetate:Pentane to yield 320 mg product.

Procedure:

126 mg of 3,5-dihydroxy-nonanedioic acid 1-tert-butyl ester 9-ethyl ester was dissolved in 16 mL of dry toluene then cooled to −10° C. in an acetone/ice bath. To this was added 4 mg of Tosic Acid. After 8 hours at −10° C., the S.M. appeared consumed. The reaction was quenched with saturated sodium bicarbonate solution and partitioned between ethyl acetate and saturated sodium bicarbonate solution. The ethyl acetate was removed in vacuo and the residue purified via flash chromatography (1:1 Ethyl Acetate:Pentane->1.5:1 Ethyl Acetate:Pentane R_(f)=0.3 in 1.5:1 E.A.:Pentane). Yield=75%

Procedure:

29 mg of 3-hydroxy-4-(6-oxo-tetrahydro-pyran-2-yl)-butyric acid tert-butyl ester was dissolved in 3 mL of dichloromethane and cooled to 0° C. To this was added 24 mg of dry 2,6-lutidine. 60 mg of TBS triflate was added followed by 50 mg of DMAP. The solution was allowed to warm to r.t. and stirred overnight. The reaction was then partitioned between ethyl acetate and saturated bicarbonate solution. The ethyl acetate was removed in vacuo and the residue purified via flash chromatography (15% ethyl acetate:pentane R_(f)=0.3) to yield the TBS ether in 72% yield.

Procedure:

To 190 mg of NaH (95%) in 80 mL of dry THF was added 1.05 g of ethyl acetoacetate in 15 mL of dry THF at 0° C. After 10 minutes, 5.4 mL of 1.5 M n-BuLi in Hexanes was added. After 10 additional minutes, 1% of the solution (˜1 mL) was taken and cooled to −78° C. in a dry flask. To this was added 15 mg of 3-(tert-butyl-dimethyl-silanyloxy)-4-(6-oxo-tetrahydro-pyran-2-yl)-butyric acid tert-butyl ester in 1 mL of dry THF. After 30 minutes, the reaction was quenched with 1N HCl and partitioned into ethyl acetate. The ethyl acetate was removed in vacuo and the residue purified by flash chromatography (30% ethyl acetate:pentane Rf=streak˜0.3).

Procedure:

5 mg of 3-(tert-butyl-dimethyl-silanyloxy)-4-[6-(3-ethoxycarbonyl-2-oxo-propyl)-6-hydroxy-tetrahydro-pyran-2-yl]-butyric acid tert-butyl ester was dissolved in 1 mL of dichloromethane and cooled to −78° C. To this was added 100 μL of TES and 50 μL of TFA. The solution was allowed to slowly warm to 0° C., at which time a lower slightly lower Rf spot cleanly formed (Rf=0.2 in 30% E.A.:Pentane). The reaction was quenched with saturated bicarbonate solution and extracted into ethyl acetate. The product was then purified via flash chromatography to yield the syn tetrahydropyran in 90% without the TBS ether.

Example 11

Compound and synthetic step references in this example correspond to the following two schemes (as opposed to the reaction schemes and formula numbers employed previously in the specification).

To a stirred solution of diisopropylamine (16.82 ml, 120 mmol) in THF (50 ml) was added n-butyllithium (48 ml, 2.5 M in hexane) dropwise at −78° C. The mixture was stirred at 0° C. for 30 min, cooled again to −78° C., and treated with methyl isobutyrate (22.5 ml, 109 mmol) slowly over 10 min. The reaction mixture was stirred for 1.5 hours at −78° C. and 1 hour at −40° C. After addition of allyl bromide (11.8 ml, 135 mmol) dissolved in THF (25 ml) the mixture was allowed to warm up to room temperature [rt]overnight. The solution was evaporated without aqueous workup. The formed solid LiBr was removed by chromatographic filtration on silica gel with ether/pentane (1:1). Fractional distillation gave 5 (12.77 g, 90%) at bp=135→150° C. as colorless liquid.

To a stirred solution of 5 (14.22 g, 101 mmol) in CCl₄ (80 ml) was added N-bromosuccinimide (20 g, 112 mmol) and dibenzoylperoxide (80 mg, 0.33 mmol) in a single portion. The reaction mixture was heated at reflux for 2 hours using an preheated oil bath (105° C.). After cooling to rt, the mixture was filtered and the residue was washed with CCl₄. The solvent was removed in vacuo and the crude material purified using flash chromatography on silica gel with EtOAc/hexane (9/1) yielding the desired allylic bromide 26 (8.34 g, 74%) as yellow oil.

6a R═OMe 6b R═OPMB 6c R═OH

To a suspension of sodium hydride (1.83 g, 45.8 mmol; 60% in mineral oil) in 100 ml anhydrous THF was added a solution of p-methoxy benzylalcohol (5.75 g, 41.6 mmol) in THF (25 ml) slowly over 15 minutes at 0° C. The mixture was stirred at rt for 45 minutes before a solution of the previously prepared allylic bromide (2.3 g, 10.4 mmol) in 30 ml THF was added over 15 min. The reaction mixture was warmed to 35° C. for 6 h. The reaction was cooled to rt and quenched with water carefully. The aqueous layer was extracted with Et₂O (2×), then neutralized with 2N HCl, and again extracted with EtOAc (4×). The combined organic layers were dried over MgSO₄ and concentrated in vacuo. Purification of the residue on silica gel (EtOAc/hexane 33%-80%) afforded 6b (2.03 g, 74%) as yellow oil.

Alternatively the reaction can be quenched with saturated NH₄Cl solution. The aqueous layer was then extracted with Et₂O (3×) and the combined layers were dried over MgSO₄ and concentrated in vacuo. The resulting oil was purified by flash chromatography (EtOAc 20%) to provide a mixture of 6a & 6b, the methyl and p-methoxybenzyl esters.

To a stirred solution of acid 6c (133 mg, 0.5 mmol) in anhydrous Et₂O (6 ml) and cooled to 0° C., was added sodium hydride (240 mg, 6.0 mmol; 60% in mineral oil) in a single portion. The mixture was stirred for 30 minutes at 0° C. and then oxalyl chloride (0.26 ml, 3.0 mmol) was added in a single portion. The resulting mixture was allowed to warm to rt and stirring was continued for 2 h. The mixture was then concentrated in vacuo and the resulting oil used without further purification.

To a stirred solution of methyl and p-methoxybenzyl esters 6a and 6b (6.56 g, 23.55 mmol) in THF (20 ml), was added N,O-dimethylhydroxylamine hydrochloride (3.68 g, 37.7 mmol), followed by dropwise addition of phenylmagnesium bromide (2M in THF, 18.25 ml, 36.5 mmol) at −20° C. To the reaction mixture was subsequently added phenylmagnesium bromide (18.25 ml, 36.5 mmol) over 45 min. Stirring was continued for 1 hour at −10° C. The reaction was quenched with saturated NH₄Cl and diluted with Et₂O. The aqueous layer was extracted with EtOAc (3×) and the combined organics were dried over MgSO₄ and concentrated in vacuo. The resulting oil was purified by flash chromatography (EtOAc/hexane 1/3) to provide Weinreb amide 8 (6.54 g, 90%) as colorless oil.

A solution of Weinreb amide 8 (5.82 g, 18.1 mmol) in THF (100 ml) was cooled to −78° C. then treated with methyllithium (1.4 M in Et₂O, 17.47 ml, 24.5 mmol). Stirring was continued for 1 hour at −78° C., and the reaction was quenched with saturated NH₄Cl, and diluted with Et₂O. The aqueous layer was extracted with EtOAc (3×) and the combined organics were dried over MgSO₄ and concentrated in vacuo. The resulting oil was purified by flash chromatography (EtOAc/hexane 1/4) to provide methyl ketone 9 (4.89 g, 99%) as a colorless oil.

To a stirred solution of diisopropylamine (0.21 ml, 1.5 mmol) in THF (3 ml) was added n-butyllithium (0.93 ml, 1.6 M in hexane) dropwise at −78° C. The mixture was allowed to warm to 0° C. and stirred for 30 min, then cooled again to −78° C., and a solution of acetone (0.11 ml, 1.5 mmol) in THF (1 ml) was added dropwise. The acetone used was dried over 4 Å molecular sieves for several days and was dried again over 4 Å molecular sieves in THF solution immediately prior to use. After stirring for 20 minutes, the mixture was treated with a solution of acid chloride 7 (0.5 mmol) in 2 ml THF and stirring was continued at −78° C. for 1 h. The reaction was quenched with saturated NH₄Cl and warmed up to rt and diluted with Et₂O. The aqueous layer was extracted with EtOAc (3×) and the combined organic layers were dried over MgSO₄ and concentrated in vacuo. Purification of the residue by flash chromatography on silica gel (EtOAc/hexane 1/2) yielded β-diketone 11 (117 mg, 77%) as an orange oil.

To a stirred solution of diisopropylamine (0.21 ml, 1.5 mmol) in THF (3 ml) was added n-butyllithium (0.93 ml, 1.6 M in hexane) dropwise at −78° C. The mixture was warmed to 0° C. and stirred for 30 minutes, then cooled again to −78° C., and treated with a solution of acetone (0.11 ml, 1.5 mmol) in THF (1 ml). The acetone used was dried over 4 Å molecular sieves for several days and was dried again over 4 Å molecular sieves in THF solution immediately prior to use. After recooling to −78° C. and stirring for 20 minutes, aldehyde 10 (164 mg, 0.5 mmol) was added dropwise and stirring was continued for 15 minutes. The reaction was quenched by addition of saturated NH₄Cl and the mixture was warmed to rt and diluted with Et₂. The mixture was diluted with Et₂O and the layers separated. The aqueous layer was then extracted with EtOAc (3×). The combined organics were washed with brine, dried over MgSO₄, and concentrated in vacuo. Flash chromatography on silica gel (EtOAc/hexane 1/2) provided 12 (169 mg, 87%). The diastereoselectivity was determined to be 81%, favoring the desired isomer, after coupling with acid chloride 7 and cyclization to pyranone 14.

From 11: To a stirred solution of diisopropylamine (1.19 ml, 8.48 mmol) in THF (13 ml) was added n-butyllithium (4.08 ml, 8.16 mmol, 2.0 M in hexane) dropwise at −78° C. The mixture was warmed to 0° C. and stirred for 30 minutes, then a solution of β-diketone 11 (2.30 g, 8.77 mmol) in THF (13 ml) was added slowly over 10 minutes. After stirring for 1 hour at 0° C., the mixture was cooled to −78° C. and aldeyde 2 (1.21 g, 3.69 mmol) was added in a single portion. Stirring was continued for 30 minutes at −78° C. and the reaction mixture was then quenched with saturated NH₄Cl solution, allowed to warm to rt, and diluted with Et₂O. The mixture was extracted with EtOAc (3×) and the combined organics were dried over MgSO₄ and concentrated in vacuo. Flash chromatography on silica gel (EtOAc/hexane 1/2) afforded the aldol 13 (1.51 g, 65%) as a orange oil. The ratio of the two diastereomers was determined after cyclization to pyranone 7, and was 1.45:1 favoring the desired isomer.

From 12: To a stirred solution of diisopropylamine (0.46 μL, 0.204 mmol) in THF (0.5 ml) was added n-butyllithium (0.128 ml, 0.204 mmol, 1.6M in hexanes) dropwise at −78° C. The mixture was warmed to 0° C. and stirred for 30 min, then cooled again to −78° C., and treated with a solution of β-hydroxy ketone 12 (42.0 mg, 0.0662 mmol) in THF, (1 ml) dropwise. After stirring for 20 minutes at −78° C. the mixture was treated with a solution of acid chloride 7 (0.5 ml, ˜0.20 mmol, prepared from 0.5 mmol acid 6c dissolved in 1 ml THF) and stirring was continued at −78° C. for 30 min. The reaction was quenched with H₂O, warmed up to rt by stirring vigorously for 30 min, and diluted with Et₂O. The aequeous layer was extracted with EtOAc (3×) and the combined organic layers were dried over MgSO₄ and concentrated in vacuo. Purification of the residue by flash chromatography yielded 13 (41.9 mg, 61%).

From 28: To distilled pyridine (1.3 ml, 16.8 mmol) in a high density polyethylene (HDPE) vial at −78° C. was added dropwise over 10 minutes a solution of 70% HF.pyridine (0.5 ml, ˜17.5 mmol HF, ˜4.4 mmol pyridine) to form a nearly equimolar solution of HF.pyridine. This solution was stored at −20° C. until needed.

To a solution of the previously prepared C23 OTBS β-diketone 28 (0.025 g, 0.033 mmol) in dry THF (2 ml) in a HDPE vial was rapidly added the HF.pyridine solution prepared above (0.25 ml) in a single portion. The resulting solution was layered with argon, sealed and stirred vigorously for 7 days at rt. The vial was unsealed, and the reaction quenched with saturated aqueous NaHCO₃ (1 ml). The reaction was diluted with ethyl acetate, the layers separated, and the aqueous phase extracted 3 times with ethyl acetate. The combined organic fractions were pooled, dried over MgSO₄ and concentrated under reduced pressure. The resulting oil was subjected to flash chromatography in 30% ethyl acetate/hexanes, which yielded β-diketo alcohol 13 (0.015 g, 0.051 mmol, 70%) as a colorless oil.

To a stirred solution of 13 (1.28 g, 2.02 mmol) in toluene (30 ml) was added p-toluene sulfonic acid (0.060 g, 0.0003 mmol) in a single portion followed by addition of 4 Å molecular sieves (1.5 g). After stirring at rt for 10 hours, the reaction was quenched with pyridine (2.0 ml, 24.7 mmol) and filtered. The resulting solution was concentrated under reduced pressure, then redissolved in dietyhl ether. This was washed with saturated NaHCO₃ and dried over MgSO₄ before the solvent was removed under reduced pressure. The resulting oil was subjected to flash chromatography in 30% ethyl acetate in hexanes, which was increased to 70% as the product began to elute from the column. This yielded 1.00 g (1.63 mmol, 81%) of 14 as a 1.54:1 mixture of diastereomers at C23 which were separable after a second chromatographic step in which 300 g of silica gel were used per 1 g of the pure diastereomers, and the material was eluted again using 30% ethyl acetate in hexanes.

In a glove bag, under positive Ar pressure, 0.9834 g (3.11 mmole) methoxy diisopinylborane was weighed into a dried flask. Dry diethyl ether (8.4 ml) was added, and the resulting solution cooled to −78° C. To this solution was added dropwise over 5 minutes a 1M solution of allyl magnesium bromide (2.8 ml, 2.8 mmol), after which the solution was allowed to come to rt over 1 hour. A portion (6.2 ml, 2.06 mmole) of the resulting borane reagent was added to a stirred solution of the aldehyde 10 (0.6546 g, 2.0 mmol) in diethyl ether (5 ml) at −78° C. over 15 minutes. The reaction was stirred at −78° C. for 1 hour, and then allowed to warm to rt over 1 hour. The resulting boronate was then cleaved by addition of 10 ml of 15% NaOH and 2 ml 30% H₂O₂. This mixture was stirred for 30 minutes, and the layers were separated. The aqueous layer was then extracted 4 times with diethyl ether. The combined organic phases were dried over MgSO₄ and concentrated under reduced pressure. The resultant oil was subjected to flash chromatography in 15% ethyl acetate/hexanes produced 15 (0.4723 g, 1.28 mmol, 64%) as a clear oil.

To a stirred solution of 15 (3.39 g, 9.19 mmol) in dry CH₂Cl₂ (80 ml) was added a 60% dispersion of NaH in mineral oil (0.551 g, 13.79 mmol) in a single portion, and the resulting solution was stirred at rt for 1 h. To this was added dimethyl aminopyridine (0.061 g, 0.5 mmol), followed by t-butyl dimethylsilyl chloride (2.216 g, 14.70 mmol). The solution was then refluxed for 16 h, after which the solution was cooled to rt and quenched with 10 ml of a saturated solution of aqueous ammonium chloride. The layers were separated, and the aqueous phase extracted 3 times with ethyl acetate. The combined organic phases were dried over MgSO₄ and concentrated under reduced pressure. The resultant oil was subjected to flash chromatography in 7.5% ethyl acetate/hexanes, yielding the TBS protected allyl alcohol 27 (3.833 g, 7.90 mmol, 86%) as a clear oil.

To a stirred solution of the previously prepared TBS allyl alcohol 27 (0.035 g, 0.072 mmol) in 2:1 THF/water (3 ml) was added N-methyl morpholine N-oxide (0.0092 g, 0.079 mmol) in one portion, followed by the rapid addition of a 2.5% solution of osmium tetroxide in isopropanol (0.090 ml, 0.07 mmol), again in one portion. The resultant solution was stirred 6 hours at rt before being stopped by the addition of excess solid sodium sulfite. The reaction mixture was then diluted with brine and ethyl acetate. The layers were separated, and the aqueous phase extracted three times with ethyl acetate. The combined organic phases were concentrated under reduced pressure. The resultant oil was redissolved in 3 ml 2:1 tetrahydrofuran/water, to which was added sodium periodate (0.052 g, 0.243 mmol). The reaction mixture was stirred 6 hours at rt before being diluted with water and ethyl acetate. The layers were separated, and the aqueous phase extracted three times with ethyl acetate, and the combined organic phases concentrated under reduced pressure. The resultant yellow oil was subjected to flash chromatography in 10% ethyl acetate/hexanes, and yielded aldehyde 16 (0.0297 g, 0.061 mmol, 85% over 2 steps) as a clear oil.

To a stirred solution of diisopropyl amine (0.352 ml, 2.51 mmol) in dry THF (25 ml) at −78° C. was added dropwise a 2.5M solution of nBuLi in hexanes (0.956 ml, 2.39 mmol). The solution was stirred for 30 minutes at 0° C. and then cooled to −78° C. A solution of methyl ketone 9 (0.592 g, 2.26 mmol) in dry THF (5 ml) was cannulated slowly into the reaction mixture over 10 minutes. The resulting solution was stirred at −78° C. for 30 minutes, warmed to rt for 2 minutes, then recooled to −78° C., after which a solution of aldehyde 16 (1.0 g, 2.05 mmol) in dry THF (3 ml) was slowly cannulated into the reaction mixture over 10 minutes. This was stirred at −78° C. for 15 minutes, then quenched with a saturated solution of aqueous ammonium chloride (25 ml). The reaction was then diluted with ethyl acetate, and the layers separated. The aqueous phase was extracted 3 times with ethyl acetate, and the combined organic phases dried with MgSO₄ and concentrated under reduced pressure. The resulting oil was subjected to flash chromatography in 10% ethyl acetate in hexanes, which was increased to 20% as the product began to elute from the column. This yielded 1.48 g (1.97 mmol, 96%) of an inconsequential mixture of diastereomers which were carried through to the next reaction.

To a stirred solution of the diastereomeric mixture of β-keto alcohols (0.1103 g, 0.147 mmol) in dry CH₂Cl₂ was added N-methyl morpholine N-oxide (0.1418 g, 1.207 mmol) and approximately 0.1 g powdered 4 Å molecular sieves. Tetrapropyl ammonium perruthenate (0.0131 g, 0.037 mmol) was added and the mixture stirred for 30 minutes. The reaction mixture was then filtered through a short plug of silica with copious quantities of ethyl acetate. The resulting solution was concentrated under reduced pressure and subjected to flash chromatography using 15% ethyl acetate in hexanes. This yielded 0.0513 g (0.069 mmol, 47%) of the desired β-diketone 28.

To a solution of pyranone 14 (0.205 g, 0.333 mmol) and cerium chloride heptahydrate (0.030 g, 0.082 mmol) in 5.5 ml methanol was added solid NaBH₄ (0.012 g, 0.33 mmol) in a single portion at −20° C. The reaction mixture was stirred for 1 hour at −20° C. and monitored by tlc. A second portion of NaBH₄ (0.012 g, 0.33 mmol) was added during this period when the reaction appeared stalled. The reaction was then quenched with 20 ml saturated aqueous NaCl, and the mixture brought to rt, filtered through a pad of Celite® and the layers separated. The separated aqueous layer was extracted four times with ethyl acetate, and the combined organics were dried over Na₂SO₄ and concentrated under reduced pressure to afford the crude allylic alcohol. This moderately stable oil was reacted further without purification.

The crude allylic alcohol was dissolved in 6 ml CH₂Cl₂/MeOH (2:1) and cooled to 0° C. before being treated with solid NaHCO₃ (0.042 g, 0.5 mmol). Purified m-chloroperoxybenzoic acid (0.046 g, 0.370 mmol) was added in a single portion and the reaction mixture was stirred for 30 minutes, then warmed to rt over a period of 15 minutes. The reaction was quenched with triethylamine (4.0 ml), stirred well for 20 minutes, diluted with 40 ml diethyl ether, and filtered through a pad of Celite®. The filtrate was concentrated under reduced pressure and the resulting oil purified using flash chromatography using (EtOAc/hexane 1/1) to give diol 17 (0.158 g, 0.238 mmol, 71%) as a colorless oil.

A solution of diol 17 (118 mg, 0.178 mmol) and 4-dimethylaminopyridine (77 mg, 0.62 mmol) in CH₂Cl₂ (3.2 ml) was cooled to −10° C. and treated with benzoyl chloride (27 μl, 0.23 mmol) dropwise via syringe. The resulting mixture was stirred at −10° C. for 30 minutes, quenched with saturated NaHCO₃ and diluted with EtOAc (20 ml). The organic layer was washed with H₂O and brine, dried over Na₂SO₄ and concentrated in vacuo to afford a crude mixture of C21 monobenzoate and 4-dimethylaminopyridine as a colorless paste, which was filtered over a plug of silica gel (EtOAc/hexane 1/2).

The filtrate was evaporated and taken up in 8 ml CH₂Cl₂ and treated with solid 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (Dess-Martin periodinane, 113 mg, 0.267 mmol) at rt. The solution was stirred for 4 hours at rt after which a second portion (113 mg, 0.267 mmol) of DMP was added. The opaque white mixture was stirred for another 1.5 hours and quenched with 4 ml saturated NaHCO₃/Na₂S₂O₃. The layers were separated and the aqueous phase was extracted with CH₂Cl₂ (2×). The combined organics were dried over Na₂SO₄ and concentrated in vacuo to provide a colorless semisolid. Flash chromatography on silica gel (EtOAc/hexane 1/3) gave the desired keto-pyranone 29 (121 mg, 89% from 17) as a colorless oil.

To a stirred solution of diisopropylamine (300 μl, 2.14 mmol) in THF (3.2 ml) was added n-butyllithium (1.25 ml, 1.6 M in hexanes, 2.00 mmol) dropwise at −78° C. The mixture was warmed to 0° C. and stirred for 30 min, then cooled again to −78° C., and a solution of ketone 18 (319 mg, 0.494 mmol) in 6.8 ml THF was added in a single portion. The solution was stirred for 30 minutes and treated with a solution of freshly distilled OHCCO₂Me (88 mg, 0.74 mmol) in 5 ml THF, kept at −78° C. for 30 minutes and quenched with 3 ml saturated NH₄Cl. The reaction mixture was brought to rt and diluted with 200 ml EtOAc. The organic layer was washed with H₂O (2×) and brine, dried over Na₂SO₄ and concentrated in vacuo. The crude residue was chromatographed on silica gel (EtOAc/hexanes 35/65) to afford the aldol product (319 mg, 88%) as an inconsequential mixture of diastereomers.

The isolated aldol product (303 mg, 0.412 mmol) and Et₃N (340 μl, 2.50 mmol) were dissolved in anhydrous CH₂Cl₂ (15 ml) and cooled to −10° C. Methanesulfonylchloride (97 μl, 1.25 mmol) was added via syringe and the solution was stirred at −10° C. for 30 minutes and warmed to rt. 5 ml saturated NaHCO₃ were added and the reaction mixture was diluted with 100 ml EtOAc. The organic layer was washed with H₂O and brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was immediately dissolved in THF (20 ml) and treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU—75 μl, 0.5 mmol) dropwise at rt. The resulting bright yellow solution was stirred at rt for 20 minutes, treated with saturated NH₄Cl and diluted with 150 ml EtOAc. The organic layer was washed with H₂O and brine, dried over Na₂SO₄ and concentrated in vacuo to afford an orange residue which was chromatographed on silica gel (20% EtOAc/hexanes) to afford exocyclic methacrylate 30 (239 mg, 81%—unseparable mixture of E/Z isomers, ratio E/Z=7:1) as a yellow oil.

To a solution of enone 19 (205 mg, 0.286 mmol) and cerium chloride heptahydrate (52 mg, 0.15 mmol) in 11 ml methanol was added solid NaBH₄ (21 mg, 0.57 mmol) in a single portion at −30° C. Rapid gas evolution subsided after 3 minutes. After an additional 30 minutes at −30° C., the reaction mixture was poured directly onto a silica gel column and the product quickly eluted with EtOAc/hexanes (3/1) to afford the alcohol as colorless oil.

Octanoic acid (93 mg, 0.64 mmol) and Et₃N (117 μl, 0.88 mmol) were dissolved in 8 ml toluene and treated with 2,4,6-trichlorobenzoylchloride (92 μl, 0.59 mmol) dropwise at rt. After 2.5 hours at rt, a toluene solution (5 ml) of freshly prepared alcohol was added gradually via syringe and stirring was continued for 1 h. The reaction mixture was quenched with 10 ml saturated NaHCO₃, diluted with EtOAc and washed successively with saturated NH₄Cl and brine. The organics were dried over Na₂SO₄, the solvent was removed in vacuo, and the residue was chromatographed on silica gel (EtOAc/hexane 1/3) to provide octanoate 20 as an colorless oil (208 mg, 86% from 19).

To a solution of 20 (2.0 mg, 0.0024 mmol) in 1 mL 1% aqueous CH₂Cl₂ was added solid 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 1.2 mg, 0.0053 mmol) at 0° C. The reaction mixture was stirred for 30 minutes, then pipetted directly onto a plug of silica gel. The product was eluted with EtOAc/hexane (1/4) and the solvent was removed in vacuo to provide the crude diol (1.1 mg). This material was dissolved in anhydrous CH₂Cl₂ and treated with manganese (IV) oxide (0.4 mg, 0.0046 mmol) at 0° C. The reaction mixture was allowed to warm to rt and then pipetted directly onto a plug of silica gel. The product was eluted with EtOAc/hexane (1/3) and the solvent was removed in vacuo. Crude aldehyde 33 (0.64 mg, 42%) was obtained as a colorless oil.

Example 12

Compound and synthetic step references in this example correspond to the following scheme (as opposed to the reaction schemes and formula numbers employed previously in the specification).

A 3-neck flask was charged with NaH (16 g, 60 wt % in mineral oil, 0.40 mol). The NaH was washed with Et₂O (3×40 mL). THF (800 mL) was added and the mixture stirred. To this suspension was added 2,2-dimethyl-1,3-propanediol 1 (40 g, 0.39 mol) in portions over 10 min. The resulting thick slurry was stirred at room temperature [rt] for 1 h. TBSCl (60.5 g, 0.40 mol) was added in one portion. The slurry thinned and was stirred at rt for 14 h. The reaction was diluted with MTBE (1.0 L) and washed with 10% aq. K₂CO₃ (700 mL, 300 mL) and brine (500 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to yield 88.4 g of a viscous liquid which was used without further purification.

A solution of 2 (40 g, ca 0.18 mol) was stirred in CH₂Cl₂ (900 mL) and cooled in an ice bath to 4° C. To this solution was added NEt₃ (76 mL, 0.54 mol). A slurry of SO₃.pyr (44 g, 0.27 mol) in DMSO (100 mL) was added in two portions 5 min apart to keep T<10° C. The reaction was stirred for 2.5 h and the ice bath removed. Stirring was continued for 6.5 h and another portion of SO₃.pyr (10 g, 63 mmol) was added as a solid. The reaction was stirred for 9 h and diluted with CH₂Cl₂ (1.0 L). The resulting solution was washed with 1 N aq. HCl (2×500 mL), satd. aq. NaHCO₃ (500 mL), and brine (500 mL). The resulting organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to 39.8 g of an orange liquid which was used without further purification.

A solution of 4-chloro-1-butanol (23 mL, 0.23 mol) in THF (230 mL) was cooled to −78° C. A solution of MeMgCl (77 mL, 3.0 M in THF, 0.23 mol) was added dropwise via addition funnel over 30 min to keep T<−60° C. The reaction was let warm to −10° C. and Mg° (6.04 g, 0.251 mol) was added followed by BrCH₂CH₂Br (0.1 mL). The reaction was heated to reflux for 14.5 h. Heating was removed, THF (220 mL) was added, and the reaction was cooled to −78° C. A solution of 3 (39 g, ca 0.18 mol) in THF (100 mL) was added dropwise via addition funnel over 40 min to keep T<−60° C. The reaction was stirred 30 min at −78° C. and the cold bath removed. MTBE (500 mL) was added when the reaction reached −30° C. followed by aq. citric acid (87 g, 0.41 mol in 500 mL H₂O). The organic layer was collected and the aqueous layer was extracted with MTBE (200 mL). The combined organic layers were washed with brine (2×400 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to a light orange oil. The oil was dissolved in EtOAc and filtered through silica. The filtrate was concentrated under reduced pressure to yield 49.7 g of a light yellow oil which was used without further purification.

To a solution of CH₂Cl₂ (800 mL) was added (COCl)₂ (46.9 mL, 0.537 mol) and the resulting solution cooled to −78° C. DMSO (75.8 mL, 1.07 mol) was added dropwise via addition funnel over 15 min to keep T<−60° C. The resulting solution was stirred for 10 min. A solution of 4 (49.7 g, ca 0.179 mol) in CH₂Cl₂ (150 mL) was added dropwise via addition funnel over 25 min to keep T<−70 C. The resulting mixture was stirred 1 h at −78° C. and NEt₃ (250 mL, 1.79 mol) was added dropwise via addition funnel over 15 min to keep T<−60° C. The cold bath was removed and the reaction was allowed to warm to −30° C. and then poured into a mixture of H₂O (500 mL) and CH₂Cl₂ (800 mL). The resulting organic layer was collected and washed with H₂O (500 mL), satd. aq. NH₄Cl (2×400 mL), satd. aq. NaHCO₃ (500 mL), and brine (500 mL). The organic layer was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure to a dark oil. Purification by flask chromatography on silica in 19:1→9:1 pet. ether:EtOAc yielded 27.81 g (54% based on 2,2-dimethyl-1,3-propanediol) of a straw yellow liquid.

A 3-neck flask was charged with powdered 4 Å mol. sieves (47.5 g), CH₂Cl₂ (600 mL), and R-BINOL (3.6 g, 12.5 mmol). To this mixture was added Ti(OiPr)₄ (1.84 mL, 6.25 mmol). The resulting orange mixture was heated to reflux for 1 h, then cooled to rt in a water bath. A solution of 5 (36.0 g, 125 mmol) in CH₂Cl₂ (60 mL) was added followed by B(OMe)₃ (16.8 mL, 150 mmol) and Bu₃SnCH₂CHCH₂ (46.5 mL, 150 mmol). The resulting mixture was stirred at rt for 42 h, then filtered through celite into saturated aqueous NaHCO₃ (200 mL). The resulting mixture was stirred for 1 h. The organic layer was collected and the aqueous layer was extracted with CH₂Cl₂ (200 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to a thick orange oil. The residue was purified by flash chromatography on silica in 9:1→4:1 pet. ether:EtOAc to yield 29.03 g, (70%) of a yellow oil.

To a solution of 6 (28.8 g, 87.6 mmol) in MePh (500 mL) was added beaded 4 Å mol. sieves (50 g) andpTsOH (1.66 g, 8.76 mmol). The mixture was stirred at rt for 18 h then filtered through basic alumina (Brockman grade I, basic, 150 mesh) and the alumina washed with pet. ether. The filtrate was concentrated under reduced pressure to 23.2 g (85%) of a clear liquid.

A mixture of 80% MMPP (25.7 g, 41.5 mmol), CH₂Cl₂ (245 mL), MeOH (122 mL), and NaHCO₃ (8.87 g) was stirred in an ice bath. A solution of 7 (21.5 g, 69.2 mmol) in CH₂Cl₂ (50 mL) was added dropwise via addition funnel over 10 min. The reaction was stirred for 10 min, and then poured into H₂O (200 mL). The organic layer was collected and the aqueous layer was extracted with CH₂Cl₂ (200 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to a thick oil which was purified by flash chromatography on silica in 19:1→9:1 pet. ether:EtOAc to yield 16.49 g (66%) of a clear oil.

To an oven-dried flask was added the major diastereomer of 8 (5.3 g, 14.8 mmol) in 75 mL CH₂Cl₂. MeCN (12 mL) was added and the reaction was cooled in an ice bath. 7.6 g 4 Å molecular sieves was added, followed by NMO (2.6 g, 22.2 mmol) and TPAP (310 mg, 0.88 mmol). The reaction was stirred for 15 min. in an ice bath, then warmed to rt. Reaction stirred overnight. Additional 4 Å molecular sieves (1.05 g), NMO (1.0 g), and TPAP (120 mg) were added. After 5 hours of stirring, at rt, the reaction was filtered through celite, with a CH₂Cl₂ was. The filtrate was concentrated in vacuo and flashed through a plug of silica, eluting with CH₂Cl₂. The fractions containing product were concentrated in vacuo to yield 4.28 g (81%) of product.

To a solution of 9 (1.72 g, 4.82 mmol) in MeOH (50 mL) was added K₂CO₃ (3.66 g, 26.5 mmol) and a solution of methyl glyoxylate (14.2 mL, ˜1.7 M, 24 mmol) in THF. The resulting mixture was stirred at rt for 55 min and then poured into a mixture of satd. aq. NH₄Cl (200 mL) and Et₂O (100 mL). The organic layer was collected and the aqueous layer was extracted with Et₂O (100 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated under reduced pressure to an orange oil which was purified by flash chromatography on silica in 19:1 pet. ether:EtOAc to yield 1.50 g (72%) of a yellow oil which solidified on standing.

To an oven-dried flask was added 10 (3.29 g, 7.71 mmol) in MeOH (130 mL). CeCl₃.7H₂O (1.44 g, 3.86 mmol) was added and the reaction was stirred until the salts dissolved. The reaction was then cooled to −30° C., and NaBH₄ (580 mg, 15 mmol) was added. The reaction was stirred for 20 min at −30° C. The reaction was loaded directly onto a silica column (250 g) and eluted with 6:1 hexanes:EtOAc. The fractions containing product were combined and washed with H₂O (2×85 mL) and brine (3×85 mL). The organic layer was then dried over Na₂SO₄, filtered, and concentrated in vacuo to yield 3.49 g of crude product which was used directly in the next reaction.

The crude alcohol was dissolved in CH₂Cl₂ (75 mL) and DMAP (1.41 g, 11.5 mmol) was added. Octanoic acid (1.83 mL, 11.5 mmol) was then added, followed by DIC (1.81 mL, 11.6 mmol). The reaction was stirred at rt for 20 h. The reaction was then diluted with EtOAc and brine. The organic layer was collected and the aqueous layer was extracted with EtOAc (1×). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated to 7.17 g of a solid/oil mixture. The crude material was purified via column chromatography (silica (300 g), hexanes:EtOAc, 19:1) to yield 3.98 g (93% over 2 steps) of pure product as a light yellow oil.

To a solution of TBS ether 12 (2.0 g, 3.61 mmol) in THF (36.1 mL) at rt was added 3.HF.Et₃N (6.0 mL, 36.1 mmol). The reaction mixture was stirred for 48 h and then diluted with ether (36 mL). The organic phase was washed with sat. aq. NaHCO₃ (2×20 mL) and brine (2×20 mL), dried over Na₂SO₄, and flashed through a plug of silica. The residue obtained after evaporation of solvent was carried forward without further purification. R_(f)=0.20 (hexane/ethyl acetate 4:1).

To a solution of the deprotected alcohol in CH₂Cl₂ (21 mL) and MeCN (2.1 mL), at rt, was added 4 Å molecular sieves (powder, 1.81 g) and NMO (1.06 g, 9.03 mmol). The mixture was cooled to 0° C. and TPAP (127 mg, 0.361 mmol) was added in one portion. The reaction mixture was stirred for 10 min. 0° C. and for 2 hrs. at rt. The reaction was filtered through celite, eluting with CH₂Cl₂, and concentrated in vacuo. The crude reaction mixture was then flashed through a plug of silica, also eluting with CH₂Cl₂, and then concentrated in vacuo. Purification via column chromatography (silica gel, 2.5% EtOAc/Petroleum Ether) revealed pure aldehyde 14 (1.20 g, 76% over 2 steps) as a yellow oil.

1-bromo-2-ethoxyethylene (690 μL, 6.5 mmol) was added to an oven-dried flask containing Et₂O (22 mL). The solution was cooled to −78° C., and t-BuLi (7.63 mL, 13 mmol, 1.7M in pentane) was added, dropwise. The reaction was stirred, at −78° C., for 30 min. Me₂Zn (3.35 mL, 6.7 mmol, 2.0M in toluene) was added, dropwise, and the reaction was stirred, at −78° C., for 30 min. A solution of aldehyde 14 (0.948 g, 2.2 mmol dissolved in Et₂O (22 mL, 0.1 mmol) was added dropwise, via syringe, and the reaction was stirred for 2 h at −78° C. The reaction was quenched with a 1.0 M solution of HCl (40 mL) and allowed to warm to rt. The mixture stirred vigorously for 19 h and was quenched with sat'd aq. NaHCO₃ (60 mL), diluted with EtOAc (35 mL) and H₂O (35 mL). The separated aqueous phase was extracted with EtOAc (3×60 mL) and the combined organic phases washed with brine (1×85 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. Purification via column chromatography (silica gel, Pet Ether→7%→10% EtOAc:Petroleum Ether) revealed pure enal 16 (905 mg, 90%) as a nearly colorless oil.

Preparation of stock solution: K₂OsO₂(OH)₄ (2.0 mg, 0.0054 mmol), (DHQD)₂PYR (12.0 mg, 0.0136 mmol), K₃Fe(CN)₆ (1.34 g, 4.07 mmol), and K₂CO₃ (563 mg, 4.07 mmol) were combined in a round-bottom flask, to which was added 6.75 mL of H₂O and 6.75 mL of tBuOH. The two-phase system was vigorously stirred at rt for 2 h.

Enal 16 (300 mg, 0.646 mmol) was cooled to 0° C., and a 6.4 mL aliquot of the stock solution was added. The yellow-orange colored reaction mixture was stirred at 0-4° C. for 60 h (in cold room). After diluting with water (50 mL) the aqueous phase was extracted with ethyl acetate (3×250 mL). The combined organic phases were dried over MgSO₄, and then filtered. The solvents were removed in vacuo. Purification via column chromatography (silica gel, hexane/ethyl acetate 1:9) yielded diol 17 (233 mg, 72%) as a 2.5:1 inseparable mixture of diastereomers.

The diol (10 mg, 0.020 mmol) was taken up in a solution of MeCN:H₂O (960 μL, 240 μL), and transferred to an oven-dried flask. pTsOH (38 mg, 0.200 mmol) was added as 0.22M solution in MeCN:H₂O (730 μL, 180 μL). The reaction was stirred overnight, at rt, and then quenched with sat. aq. NaHCO₃ (˜6 mL). The aqueous phase was extracted with EtOAc (×3). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude product was carried forward without further purification.

A 0.5M stock solution of 1:3 TBSCl:imidazole was prepared by dissolving TBSCl (754 mg, 5.0 mmol) and imidazole (1.02 g, 15.0 mmol) in CH₂Cl₂ (10.0 mL). A total of 9 eq. of this stock solution were added to the reaction over 6 hrs. (added in 3 eq. aliquots, 2 hrs. apart). Reaction was quenched with sat. aq. NH₄Cl and the aqueous phase was extracted with EtOAc (×3 mL). The combined organic phases were dried over Na₂SO₄, filtered, and concentrated in vacuo. Purification via column chromatography (silica gel, Hexanes:EtOAc, 75:25) revealed pure product (5.6 mg, 47% over 2 steps).

Example 13

Compound and synthetic step references in this example correspond to the following scheme (as opposed to the reaction schemes and formula numbers employed previously in the specification).

To a solution of acid 31 (28 mg, 0.05 mmol) in toluene (1.11 mL) was added NEt₃ (14 μL, 0.1 mmol) and 2,4,6-trichlorobenzoyl chloride (8.3 μL, 0.05 mmol) at rt and the mixture was stirred for 1 h. To this reaction mixture was added a solution of the alcohol 19 (15 mg, 0.025 mmol) and DMAP (15 mg, 0.125 mmol) in toluene (1.1 mL) at rt. The reaction mixture was stirred for 1 h and then directly poured onto a column (silica gel, hexane/ethyl acetate 85:15) and diluted with this solvent mixture to afford 24 mg (87%) of ester 32 as a colorless oil.

To a solution of aldehyde 32 (20 mg, 0.018 mmol) in THF (5 mL) at rt in a plastic vial was added 70% HF/pyridine (968 μL, excess) dropwise and the yellow reaction mixture was stirred for 1.5 h. The reaction was quenched by addition of sat. aq. NaHCO₃ solution (12.5 mL) and H₂O (7.5 mL). The biphasic mixture was extracted with ethyl acetate (4×12 mL). The combined organic phases were dried over Na₂SO₄. After filtration and evacuation of solvents the residue was purified by column chromatography (silica gel, hexane/ethyl acetate 1:4) to yield 11 mg (82%) of Formula 33 as a white solid.

Example 14

Analogues 19B.1, 19B.2, 19B.3, and 19B.4 were synthesized as described in Reaction Schemes 17, 18, and 19. All of the analogues were assayed against a rat brain PKC isozyme mixture and their binding affinities obtained. The results as shown in Table 4, indicate that lipophilic groups substituted at the para position are well tolerated.

TABLE 4 Analogue R Binding Affinity in nM 19B.0 R is H 2.3 18C R is BR 1.6 19B.1 R is phenyl 3.1 19B.2 R is —(CH₂)₃O(CH₂)₂OCH₂CH₃ 3.8 19B.3 R is —C═C(CH₂)₅CH₃ 4.6 18B.4 R is —C═C(CH₂)₁₁CH₃ 30

Example 15

Compound 20B was produced as described in Reaction Scheme 20 in 86% yield after chromatography.

R_(f): 0.36 (EtOAc:petroleum ether 4:1), one spot, brown (p-anisaldehyde stain). [α]_(D): +107.3° (c=0.15, CDCl₃).

¹H NMR: (600 MHz, CDCl₃) δ 6.09 (d, J=15.6 Hz, 1H, C17-H), 5.75 (s, 1H, C20-H), 5.41 (dd, J=16.2, 7.2 Hz, 1H, C16-H), 5.26-5.24 (m, 1H, C22-H), 5.24-5.22 (m, 1H, C25-H), 5.13 (d, J=7.2 Hz, 1H, C15-H), 4.83 (s, 1H, C19-OH), 4.42-4.37 (m, 1H, C23-H), 4.27-4.19 (m, 2H, C3-H, C3-OH), 4.11-4.06 (m, 1H, C9-H), 3.94-3.86 (m, 2H, C5-H, C26-OH), 3.84-3.77 (m, 1H, C26-H), 3.66 (s, 3H, —OCH₃) 3.56-3.50 (m, 1H, C13-H), 3.47-3.41 (m, 1H, C13-H), 3.03 (d, J=16.8 Hz, 1H, C34-H), 2.94 (d, J=16.8 Hz, 1H), 2.61-2.51 (m, 2H, C24-H), 2.38-2.27 (m, 2H), 2.01-1.95 (m, 2H), 1.91-1.74 (m, 5H), 1.69-1.57 (m, 3H), 1.52-1.43 (m, 4H), 1.38-1.21 (m, 14H, C27-H₃), 1.19 (s, 3H, C18-CH₃), 0.99 (s, 3H, C18-CH₃), 0.87 (t, J=7.2 Hz, 3H, octanoate-CH₃) ppm. ¹³C-NMR: (125 MHz, CDCl₃) δ173.4, 172.2, 171.4, 143.5, 131.0, 127.9, 125.2 102.6, 98.5, 78.9, 76.4, 75.7, 73.8, 70.4, 69.4 (2C), 66.3, 63.7, 51.9, 45.0, 42.8, 42.7, 40.5, 39.0, 34.9, 34.6, 32.5, 31.6, 31.43, 31.36, 29.1, 28.9, 24.6, 24.2, 23.0, 22.6, 20.3, 18.9, 14.1 ppm.

IR (FTIR, film) v=3461, 3368, 2930, 2856, 1736, 1459, 1432, 1407, 1379, 1360, 1296, 1281, 1250, 1200, 1158, 1137, 1106, 1077, 1049, 1013, 975, 909, 895, 857, 806, 779 732, 589 cm⁻¹

HRMS: m/z=761.4069 (M+Na)⁺. Calculated for C₃₉H₆₂O₁₃Na: 761.4083.

All references cited herein are hereby incorporated by reference. Although the invention has been described with respect to specific embodiments and examples, it will be appreciated that various changes and modifications may be made without departing from the spirit of the invention. 

What is claimed is:
 1. A compound of Formula I

wherein: X is S, N(optionally substituted alkyl), N(optionally substituted alkenyl), N(optionally substituted alkynyl), N(optionally substituted aryl), N(optionally substituted heteroaryl), N(optionally substituted aralkyl), or N(optionally substituted heteroaralkyl), R³ is H or OH; R⁷ is selected from the group consisting of optionally substituted lower alkyl, optionally substituted alkenyl, hydroxyl, amino, optionally substituted alkylamino, ═O, optionally substituted acylamino, OC(O)NR′R′, OC(O)OR′, OC(O)R′, and substituted acyloxy; p is 1, 2, 3, or 4; R²⁰ is H, OH, or -T-U—V—R′ where: T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent; R²¹ is ═CR^(a)R^(b) or R²¹ represents independent moieties R^(c) and R^(d) where: R^(a) and R^(b) are independently H, CO₂R′, CONR^(c)R^(d) or R′; R^(c) and R^(d) are independently H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3; R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof; or wherein: X is O, R³ is H or OH; R⁷ is selected from the group consisting of optionally substituted alkenyl, OC(O)NR′R′, and OC(O)OR′; p is 1, 2, 3, or 4; R²⁰ is H, OH, or -T-U—V—R′ where: T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent; R²¹ is ═CR^(a)R^(b) or R²¹ represents independent moities R^(c) and R^(d) where: R^(a) and R^(b) are independently H, CO₂R′, CONR^(c)R^(d) or R′; R^(c) and R^(d) are independently H, alkyl alkenyl, or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3; R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof.
 2. The compound of claim 1 having the stereochemical configuration of Formula IA:


3. The compound of claim 1 wherein R³ is OH.
 4. The compound of claim 1 wherein R²⁶ is H.
 5. A method of treating a mammal suffering from a disorder mediated by protein kinase C activity comprising administering to said mammal a therapeutically effective amount of the compound of claim
 1. 6. A compound of Formula II

wherein: R⁷ is selected from the group consisting of optionally substituted alkenyl, OC(O)NR′R′, and OC(O)OR′; p is 1, 2, 3, or 4; R²⁰ is H, OH, or -T-U—V—R′ where: T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent; R²¹ is ═CR^(a)R^(b) or R²¹ represents independent moieties R^(c) and R^(d) where: R^(a) and R^(b) are independently H, CO₂R′, CONR^(c)R^(d) or R′; R^(c) and R^(d) are independently H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3; R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted aralkenyl, and optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof.
 7. The compound of claim 6, having the stereochemical configuration of Formula IIA:


8. The compound of claim 6 wherein R²⁶ is H.
 9. A method of treating a mammal suffering from a disorder mediated by protein kinase C activity comprising administering to said mammal a therapeutically effective amount of the compound of claim
 6. 10. The compound of claim 6, selected from the group consisting of:

and their pharmaceutically acceptable salts thereof.
 11. A compound of Formula III:

wherein: R³ is H or OH; R⁸ is selected from the group consisting of H, OH, R′, —(CH₂)_(n)O₂CR′, and —(CH₂)_(n)O₂C-haloalkyl; n is 0, 12, 3, 4, or 5; R⁹ is H or OH; R²⁰ is H, OH, or -T-U—V—R′ where: T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent; R²¹ is ═CR^(a)R^(b) or R²¹ represents independent moieties R^(c) and R^(d) where: R^(a) and R^(b) are independently H, CO₂R′, CONR^(c)R^(d) or R′; R^(c) and R^(d) are independently H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3; R²⁶ is H or R′; and R′ is independently selected from the group consisting of substituted alkenyl, substituted alkynyl, substituted aryl, substituted heteroaryl, substituted aralkyl, substituted aralkenyl, and substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof, or a compound selected from the group consisting of:

and theft pharmaceutically acceptable salts thereof; with the proviso that the compound is not


12. The compound of claim 11 having the stereochemical configuration of Formula IIIA:


13. The compound of claim 11 wherein R³ is OH.
 14. The compound of claim 11 wherien R²⁶ is H.
 15. The compound of claim 11, selected from the group consisting of

and their pharmaceutically acceptable salts thereof.
 16. A method of treating a mammal suffering from a disorder mediated by protein kinase C activity comprising administering to said mammal a therapeutically effective amount of the compound of claim
 11. 17. A compound of Formula IV

R³ is H or OH; R⁷ is selected from the group consisting of optionally substituted alkenyl, OC(O)NR′R′, and OC(O)OR′; p is 0, 1, 2, 3, or 4; R²⁰ is H, OH, or -T-U—V—R′ where: T is selected from —O—, —S—, —N(H)— or —N(Me)—; U is absent or is selected from —C(O)—, —C(S)—, —S(O)— or —S(O)₂—; and V is absent or is selected from —O—, —S—, —N(H)— or —N(Me)—, provided that V is absent when U is absent; R²¹ is H, alkyl, alkenyl or alkynyl, or (CH₂)_(n)CO₂R′ where n is 1, 2 or 3; R²⁶ is H or R′; and R′ is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted aralkenyl, and optionally substituted heteroaralkyl, and their pharmaceutically acceptable salts thereof.
 18. The compound of claim 17 having the stereochemical configuration of Formula IVA:


19. The compound of claim 17 wherein R³ is OH.
 20. The compound of claim 17 wherein R²⁶ is H.
 21. The compound of claim 17 with the following structure;


22. A method of treating a mammal suffering from a disorder mediated by protein kinase C activity comprising administering to said mammal a therapeutically effective amount of the compound of claim
 17. 23. A method of manufacture of an analogue of bryostatin of Formula I comprising the steps of: a. converting an acid of a compound of Formula G-1 to a mixed anhydride; b. reacting said mixed anhydride of said compound of Formula G-1 with an alcohol of a compound of Formula G-2; c. transacetylizing a ketal of said compound of Formula G-1 with an aldehyde of said compound of Formula G-2; and d. removing protecting groups to yield said analogue of bryostatin of Formula II


24. A method of manufacture of an analogue of bryostatin of Formula IV comprising the step of: irradiating a compound of Formula G-3 to yield a compound of Formula IV


25. A method of treating a mammal suffering from a hyperproliferative cellular disorder or an immune-related disorder comprising administering a therapeutically effective amount of a compound of Formula I, II, III or IV, of claims 1, 6, 11 and 17, respectively or its pharmaceutically acceptable salts, to said mammal.
 26. The method of claim 25 wherein the hyperproliferative cellular disorders or immune related disorders are selected from the group consisting of tumors and cancers; unwanted angiogenesis, psoriasis, blood vessel proliferation disorders, fibrotic disorders, autoimmune disorders, disorders brought about by abnormal proliferation of mesangiai cells. 